Interferon-like molecules and uses thereof

ABSTRACT

The present invention provides Interferon-Like (IFN-L) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing IFN-L polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with IFN-L polypeptides.

FIELD OF THE INVENTION

[0001] The present invention relates to Interferon-Like (IFN-L)polypeptides and nucleic acid molecules encoding the same. The inventionalso relates to selective binding agents, vectors, host cells, andmethods for producing IFN-L polypeptides. The invention further relatesto pharmaceutical compositions and methods for the diagnosis, treatment,amelioration, and/or prevention of diseases, disorders, and conditionsassociated with IFN-L polypeptides.

BACKGROUND OF THE INVENTION

[0002] Technical advances in the identification, cloning, expression,and manipulation of nucleic acid molecules and the deciphering of thehuman genome have greatly accelerated the discovery of noveltherapeutics. Rapid nucleic acid sequencing techniques can now generatesequence information at unprecedented rates and, coupled withcomputational analyses, allow the assembly of overlapping sequences intopartial and entire genomes and the identification ofpolypeptide-encoding regions. A comparison of a predicted amino acidsequence against a database compilation of known amino acid sequencesallows one to determine the extent of homology to previously identifiedsequences and/or structural landmarks. The cloning and expression of apolypeptide-encoding region of a nucleic acid molecule provides apolypeptide product for structural and functional analyses. Themanipulation of nucleic acid molecules and encoded polypeptides mayconfer advantageous properties on a product for use as a therapeutic.

[0003] In spite of the significant technical advances in genome researchover the past decade, the potential for the development of noveltherapeutics based on the human genome is still largely unrealized. Manygenes encoding potentially beneficial polypeptide therapeutics or thoseencoding polypeptides, which may act as “targets” for therapeuticmolecules, have still not been identified.

[0004] Accordingly, it is an object of the invention to identify novelpolypeptides, and nucleic acid molecules encoding the same, which havediagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION

[0005] The present invention relates to novel IFN-L nucleic acidmolecules and encoded polypeptides.

[0006] The invention provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0007] (a) the nucleotide sequence as set forth in either SEQ ID NO: 1or SEQ ID NO: 4;

[0008] (b) the nucleotide sequence of the DNA insert in ATCC Deposit No.PTA-976;

[0009] (c) a nucleotide sequence encoding the polypeptide as set forthin either SEQ ID NO: 2 or SEQ ID NO: 5;

[0010] (d) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(c); and

[0011] (e) a nucleotide sequence complementary to any of (a)-(c).

[0012] The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0013] (a) a nucleotide sequence encoding a polypeptide which is atleast about 70 percent identical to the polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5, wherein the encoded polypeptide hasan activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQID NO: 5;

[0014] (b) a nucleotide sequence encoding an allelic variant or splicevariant of the nucleotide sequence as set forth in either SEQ ID NO: 1or SEQ ID NO: 4, the nucleotide sequence of the DNA insert in ATCCDeposit No. PTA-976, or (a);

[0015] (c) a region of the nucleotide sequence of either SEQ ID NO: 1 orSEQ ID NO: 4, the DNA insert in ATCC Deposit No. PTA-976, (a), or (b)encoding a polypeptide fragment of at least about 25 amino acidresidues, wherein the polypeptide fragment has an activity of theencoded polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5,or is antigenic;

[0016] (d) a region of the nucleotide sequence of either SEQ ID NO: 1 orSEQ ID NO: 4, the DNA insert in ATCC Deposit No. PTA-976, or any of(a)-(c) comprising a fragment of at least about 16 nucleotides;

[0017] (e) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(d); and

[0018] (f) a nucleotide sequence complementary to any of (a)-(d).

[0019] The invention further provides for an isolated nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of:

[0020] (a) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative aminoacid substitution, wherein the encoded polypeptide has an activity ofthe polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0021] (b) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acidinsertion, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0022] (c) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino aciddeletion, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0023] (d) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 which has a C- and/or N-terminaltruncation, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0024] (e) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modificationselected from the group consisting of amino acid substitutions, aminoacid insertions, amino acid deletions, C-terminal truncation, andN-terminal truncation, wherein the encoded polypeptide has an activityof the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0025] (f) a nucleotide sequence of any of (a)-(e) comprising a fragmentof at least about 16 nucleotides;

[0026] (g) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(f); and

[0027] (h) a nucleotide sequence complementary to any of (a)-(e).

[0028] The present invention provides for an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:

[0029] (a) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5; and

[0030] (b) the amino acid sequence encoded by the DNA insert in ATCCDeposit No. PTA-976.

[0031] The invention also provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0032] (a) the amino acid sequence as set forth in either SEQ ID NO: 3or SEQ ID NO: 6, optionally further comprising an amino-terminalmethionine;

[0033] (b) an amino acid sequence for an ortholog of either SEQ ID NO: 2or SEQ ID NO: 5;

[0034] (c) an amino acid sequence which is at least about 70 percentidentical to the amino acid sequence of either SEQ ID NO: 2 or SEQ IDNO: 5, wherein the polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0035] (d) a fragment of the amino acid sequence set forth in either SEQID NO: 2 or SEQ ID NO: 5 comprising at least about 25 amino acidresidues, wherein the fragment has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic; and

[0036] (e) an amino acid sequence for an allelic variant or splicevariant of the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5, the amino acid sequence encoded by the DNA insert inATCC Deposit No. PTA-976, or any of (a)-(c).

[0037] The invention further provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0038] (a) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5 with at least one conservative amino acid substitution,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5;

[0039] (b) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5 with at least one amino acid insertion, wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5;

[0040] (c) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5 with at least one amino acid deletion, wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5;

[0041] (d) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5 which has a C- and/or N-terminal truncation, wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5; and

[0042] (e) the amino acid sequence as set forth in either SEQ ID NO: 2or SEQ ID NO: 5 with at least one modification selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the polypeptide has an activity of the polypeptide set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5.

[0043] Also provided are fusion polypeptides comprising IFN-L amino acidsequences.

[0044] The present invention also provides for an expression vectorcomprising the isolated nucleic acid molecules as set forth herein,recombinant host cells comprising the recombinant nucleic acid moleculesas set forth herein, and a method of producing an IFN-L polypeptidecomprising culturing the host cells and optionally isolating thepolypeptide so produced.

[0045] A transgenic non-human animal comprising a nucleic acid moleculeencoding an IFN-L polypeptide is also encompassed by the invention. TheIFN-L nucleic acid molecules are introduced into the animal in a mannerthat allows expression and increased levels of an IFN-L polypeptide,which may include increased circulating levels. Alternatively, the IFN-Lnucleic acid molecules are introduced into the animal in a manner thatprevents expression of endogenous IFN-L polypeptide (i.e., generates atransgenic animal possessing an IFN-L polypeptide gene knockout). Thetransgenic non-human animal is preferably a mammal, and more preferablya rodent, such as a rat or a mouse.

[0046] Also provided are derivatives of the IFN-L polypeptides of thepresent invention.

[0047] Additionally provided are selective binding agents such asantibodies and peptides capable of specifically binding the IFN-Lpolypeptides of the invention. Such antibodies and peptides may beagonistic or antagonistic.

[0048] Pharmaceutical compositions comprising the nucleotides,polypeptides, or selective binding agents of the invention and one ormore pharmaceutically acceptable formulation agents are also encompassedby the invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules, and selective bindingagents.

[0049] The IFN-L polypeptides and nucleic acid molecules of the presentinvention may be used to treat, prevent, ameliorate, and/or detectdiseases and disorders, including those recited herein.

[0050] The present invention also provides a method of assaying testmolecules to identify a test molecule that binds to an IFN-Lpolypeptide. The method comprises contacting an IFN-L polypeptide with atest molecule to determine the extent of binding of the test molecule tothe polypeptide. The method further comprises determining whether suchtest molecules are agonists or antagonists of an IFN-L polypeptide. Thepresent invention further provides a method of testing the impact ofmolecules on the expression of IFN-L polypeptide or on the activity ofIFN-L polypeptide.

[0051] Methods of regulating expression and modulating (i.e., increasingor decreasing) levels of an IFN-L polypeptide are also encompassed bythe invention. One method comprises administering to an animal a nucleicacid molecule encoding an IFN-L polypeptide. In another method, anucleic acid molecule comprising elements that regulate or modulate theexpression of an IFN-L polypeptide may be administered. Examples ofthese methods include gene therapy, cell therapy, and anti-sense therapyas further described herein.

[0052] In another aspect of the present invention, the IFN-Lpolypeptides may be used for identifying receptors thereof (“IFN-Lpolypeptide receptors”). Various forms of “expression cloning” have beenextensively used to clone receptors for protein ligands. See, e.g.,Simonsen and Lodish, 1994, Trends Pharmacol. Sci. 15:437-41 andTartaglia et al., 1995, Cell 83:1263-71. The isolation of an IFN-Lpolypeptide receptor is useful for identifying or developing novelagonists and antagonists of the IFN-L polypeptide signaling pathway.Such agonists and antagonists include soluble IFN-L polypeptidereceptors, anti-IFN-L polypeptide receptor-selective binding agents(such as antibodies and derivatives thereof), small molecules, andantisense oligonucleotides, any of which can be used for treating one ormore disease or disorder, including those disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIGS. 1A-1B illustrate the nucleotide sequence of the rat IFN-Lgene (SEQ ID NO: 1) and the deduced amino acid sequence of rat IFN-Lpolypeptide (SEQ ID NO: 2). The predicted signal peptide is indicated(underlined);

[0054] FIGS. 2A-2B illustrate the nucleotide sequence of the human IFN-Lgene (SEQ ID NO: 4) and the deduced amino acid sequence of human IFN-Lpolypeptide (SEQ ID NO: 5). The predicted signal peptide is indicated(underlined);

[0055]FIG. 3 illustrates the amino acid sequence alignment of humanIFN-L polypeptide (huIFN-L; SEQ ID NO: 5), human IFN-β (huIFN-β; SEQ IDNO: 7), rat IFN-L polypeptide (raIFN-L; SEQ ID NO: 2), and those aminoacid positions which share some similarity (cons);

[0056]FIG. 4 illustrates the nucleotide sequence of the Nde I-Bam HIpAMG21 insert (SEQ ID NO: 8) of Amgen strain #3729 and the predictedamino acid sequence (SEQ ID NO: 9) encoded by this insert;

[0057]FIG. 5 illustrates the nucleotide sequence of the Nde I-Bam HIpAMG21 insert (SEQ ID NO: 10) of Amgen strain #3858 and the predictedamino acid sequence (SEQ ID NO: 11) encoded by this insert;

[0058]FIG. 6 illustrates the nucleotide sequence of the Xba I-Bam HIpAMG21 insert (SEQ ID NO: 12) of Amgen strain #4047 and the predictedamino acid sequence (SEQ ID NO: 13) encoded by this insert;

[0059]FIG. 7 illustrates the nucleotide sequence of the Xba I-Bam HIpAMG21 insert (SEQ ID NO: 14) of Amgen strain #3969 and the predictedamino acid sequence (SEQ ID NO: 15) encoded by this insert;

[0060]FIG. 8 illustrates the nucleotide sequence of the Nde I-Bam HIpAMG21 insert (SEQ ID NO: 16) of Amgen strain #4182 and the predictedamino acid sequence (SEQ ID NO: 17) encoded by this insert.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The section headings used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed. All references cited in this application are expresslyincorporated by reference herein.

[0062] Definitions

[0063] The terms “IFN-L gene” or “IFN-L nucleic acid molecule” or “IFN-Lpolynucleotide” refer to a nucleic acid molecule comprising orconsisting of a nucleotide sequence as set forth in either SEQ ID NO: 1or SEQ ID NO: 4, a nucleotide sequence encoding the polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5, a nucleotide sequence ofthe DNA insert in ATCC Deposit No.

[0064] PTA-976, and nucleic acid molecules as defined herein.

[0065] The term “IFN-L polypeptide allelic variant” refers to one ofseveral possible naturally occurring alternate forms of a gene occupyinga given locus on a chromosome of an organism or a population oforganisms.

[0066] The term “IFN-L polypeptide splice variant” refers to a nucleicacid molecule, usually RNA, which is generated by alternative processingof intron sequences in an RNA transcript of IFN-L polypeptide amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0067] The term “isolated nucleic acid molecule” refers to a nucleicacid molecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates, or other materialswith which it is naturally found when total nucleic acid is isolatedfrom the source cells, (2) is not linked to all or a portion of apolynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

[0068] The term “nucleic acid sequence” or “nucleic acid molecule”refers to a DNA or RNA sequence. The term encompasses molecules formedfrom any of the known base analogs of DNA and RNA such as, but notlimited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0069] The term “vector” is used to refer to any molecule (e.g. nucleicacid, plasmid, or virus) used to transfer coding information to a hostcell.

[0070] The term “expression vector” refers to a vector that is suitablefor transformation of a host cell and contains nucleic acid sequencesthat direct and/or control the expression of inserted heterologousnucleic acid sequences. Expression includes, but is not limited to,processes such as transcription, translation, and RNA splicing, ifintrons are present.

[0071] The term “operably linked” is used herein to refer to anarrangement of flanking sequences wherein the flanking sequences sodescribed are configured or assembled so as to perform their usualfunction. Thus, a flanking sequence operably linked to a coding sequencemay be capable of effecting the replication, transcription and/ortranslation of the coding sequence. For example, a coding sequence isoperably linked to a promoter when the promoter is capable of directingtranscription of that coding sequence. A flanking sequence need not becontiguous with the coding sequence, so long as it functions correctly.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

[0072] The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

[0073] The term “IFN-L polypeptide” refers to a polypeptide comprisingthe amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 andrelated polypeptides. Related polypeptides include IFN-L polypeptidefragments, IFN-L polypeptide orthologs, IFN-L polypeptide variants, andIFN-L polypeptide derivatives, which possess at least one activity ofthe polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.IFN-L polypeptides may be mature polypeptides, as defined herein, andmay or may not have an amino-terminal methionine residue, depending onthe method by which they are prepared.

[0074] The term “IFN-L polypeptide fragment” refers to a polypeptidethat comprises a truncation at the amino-terminus (with or without aleader sequence) and/or a truncation at the carboxyl-terminus of thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Theterm “IFN-L polypeptide fragment” also refers to amino-terminal and/orcarboxyl-terminal truncations of IFN-L polypeptide orthologs, IFN-Lpolypeptide derivatives, or IFN-L polypeptide variants, or toamino-terminal and/or carboxyl-terminal truncations of the polypeptidesencoded by IFN-L polypeptide allelic variants or IFN-L polypeptidesplice variants. IFN-L polypeptide fragments may result from alternativeRNA splicing or from in vivo protease activity. Membrane-bound forms ofan IFN-L polypeptide are also contemplated by the present invention. Inpreferred embodiments, truncations and/or deletions comprise about 10amino acids, or about 20 amino acids, or about 50 amino acids, or about75 amino acids, or about 100 amino acids, or more than about 100 aminoacids. The polypeptide fragments so produced will comprise about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids. Such IFN-L polypeptide fragments may optionally comprise anamino-terminal methionine residue. It will be appreciated that suchfragments can be used, for example, to generate antibodies to IFN-Lpolypeptides.

[0075] The term “IFN-L polypeptide ortholog” refers to a polypeptidefrom another species that corresponds to IFN-L polypeptide amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Forexample, mouse and human IFN-L polypeptides are considered orthologs ofeach other.

[0076] The term “IFN-L polypeptide variants” refers to IFN-Lpolypeptides comprising amino acid sequences having one or more aminoacid sequence substitutions, deletions (such as internal deletionsand/or IFN-L polypeptide fragments), and/or additions (such as internaladditions and/or IFN-L fusion polypeptides) as compared to the IFN-Lpolypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQID NO: 5 (with or without a leader sequence). Variants may be naturallyoccurring (e.g., IFN-L polypeptide allelic variants, IFN-L polypeptideorthologs, and IFN-L polypeptide splice variants) or artificiallyconstructed. Such IFN-L polypeptide variants may be prepared from thecorresponding nucleic acid molecules having a DNA sequence that variesaccordingly from the DNA sequence as set forth in either SEQ ID NO: 1 orSEQ ID NO: 4. In preferred embodiments, the variants have from 1 to 3,or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, orfrom 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, ormore than 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, ornon-conservative, or any combination thereof.

[0077] The term “IFN-L polypeptide derivatives” refers to thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, IFN-Lpolypeptide fragments, IFN-L polypeptide orthologs, or IFN-L polypeptidevariants, as defined herein, that have been chemically modified. Theterm “IFN-L polypeptide derivatives” also refers to the polypeptidesencoded by IFN-L polypeptide allelic variants or IFN-L polypeptidesplice variants, as defined herein, that have been chemically modified.

[0078] The term “mature IFN-L polypeptide” refers to an IFN-Lpolypeptide lacking a leader sequence. A mature IFN-L polypeptide mayalso include other modifications such as proteolytic processing of theamino-terminus (with or without a leader sequence) and/or thecarboxyl-terminus, cleavage of a smaller polypeptide from a largerprecursor, N-linked and/or O-linked glycosylation, and the like.Exemplary mature IFN-L polypeptides are depicted by the amino acidsequences of SEQ ID NO: 3 and SEQ ID NO: 6.

[0079] The term “IFN-L fusion polypeptide” refers to a fusion of one ormore amino acids (such as a heterologous protein or peptide) at theamino- or carboxyl-terminus of the polypeptide as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5, IFN-L polypeptide fragments, IFN-Lpolypeptide orthologs, IFN-L polypeptide variants, or IFN-L derivatives,as defined herein. The term “IFN-L fusion polypeptide” also refers to afusion of one or more amino acids at the amino- or carboxyl-terminus ofthe polypeptide encoded by IFN-L polypeptide allelic variants or IFN-Lpolypeptide splice variants, as defined herein.

[0080] The term “biologically active IFN-L polypeptides” refers to IFN-Lpolypeptides having at least one activity characteristic of thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5. In addition, an IFN-L polypeptide may be active as animmunogen; that is, the IFN-L polypeptide contains at least one epitopeto which antibodies may be raised.

[0081] The term “isolated polypeptide” refers to a polypeptide of thepresent invention that (1) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is naturally found when isolated from the source cell, (2)is not linked (by covalent or noncovalent interaction) to all or aportion of a polypeptide to which the “isolated polypeptide” is linkedin nature, (3) is operably linked (by covalent or noncovalentinteraction) to a polypeptide with which it is not linked in nature, or(4) does not occur in nature. Preferably, the isolated polypeptide issubstantially free from any other contaminating polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its therapeutic, diagnostic, prophylactic or researchuse.

[0082] The term “identity,” as known in the art, refers to arelationship between the sequences of two or more polypeptide moleculesor two or more nucleic acid molecules, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or polypeptides, as the casemay be, as determined by the match between strings of two or morenucleotide or two or more amino acid sequences. “Identity” measures thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

[0083] The term “similarity” is a related concept, but in contrast to“identity,” “similarity” refers to a measure of relatedness whichincludes both identical matches and conservative substitution matches.If two polypeptide sequences have, for example, 10/20 identical aminoacids, and the remainder are all non-conservative substitutions, thenthe percent identity and similarity would both be 50%. If in the sameexample, there are five more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the percent similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

[0084] The term “naturally occurring” or “native” when used inconnection with biological materials such as nucleic acid molecules,polypeptides, host cells, and the like, refers to materials which arefound in nature and are not manipulated by man. Similarly,“non-naturally occurring” or “non-native” as used herein refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man.

[0085] The terms “effective amount” and “therapeutically effectiveamount” each refer to the amount of an IFN-L polypeptide or IFN-Lnucleic acid molecule used to support an observable level of one or morebiological activities of the IFN-L polypeptides as set forth herein.

[0086] The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers to one ormore formulation materials suitable for accomplishing or enhancing thedelivery of the IFN-L polypeptide, IFN-L nucleic acid molecule, or IFN-Lselective binding agent as a pharmaceutical composition.

[0087] The term “antigen” refers to a molecule or a portion of amolecule capable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

[0088] The term “selective binding agent” refers to a molecule ormolecules having specificity for an IFN-L polypeptide. As used herein,the terms, “specific” and “specificity” refer to the ability of theselective binding agents to bind to human IFN-L polypeptides and not tobind to human non-IFN-L polypeptides. It will be appreciated, however,that the selective binding agents may also bind orthologs of thepolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, thatis, interspecies versions thereof, such as mouse and rat IFN-Lpolypeptides.

[0089] The term “transduction” is used to refer to the transfer of genesfrom one bacterium to another, usually by a phage. “Transduction” alsorefers to the acquisition and transfer of eukaryotic cellular sequencesby retroviruses.

[0090] The term “transfection” is used to refer to the uptake of foreignor exogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratories, 1989); Davis et al., Basic Methods in Molecular Biology(Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques canbe used to introduce one or more exogenous DNA moieties into suitablehost cells.

[0091] The term “transformation” as used herein refers to a change in acell's genetic characteristics, and a cell has been transformed when ithas been modified to contain a new DNA. For example, a cell istransformed where it is genetically modified from its native state.Following transfection or transduction, the transforming DNA mayrecombine with that of the cell by physically integrating into achromosome of the cell, may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been stably transformed when theDNA is replicated with the division of the cell.

[0092] Relatedness of Nucleic Acid Molecules and/or Polypeptides

[0093] It is understood that related nucleic acid molecules includeallelic or splice variants of the nucleic acid molecule of either SEQ IDNO: 1 or SEQ ID NO: 4, and include sequences which are complementary toany of the above nucleotide sequences. Related nucleic acid moleculesalso include a nucleotide sequence encoding a polypeptide comprising orconsisting essentially of a substitution, modification, addition and/ordeletion of one or more amino acid residues compared to the polypeptidein either SEQ ID NO: 2 or SEQ ID NO: 5. Such related IFN-L polypeptidesmay comprise, for example, an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites or an addition and/or adeletion of one or more cysteine residues.

[0094] Related nucleic acid molecules also include fragments of IFN-Lnucleic acid molecules which encode a polypeptide of at least about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids, or more than 200 amino acid residues of the IFN-Lpolypeptide of either SEQ ID NO: 2 or SEQ ID NO:5.

[0095] In addition, related IFN-L nucleic acid molecules also includethose molecules which comprise nucleotide sequences which hybridizeunder moderately or highly stringent conditions as defined herein withthe fully complementary sequence of the IFN-L nucleic acid molecule ofeither SEQ ID NO: 1 or SEQ ID NO: 4, or of a molecule encoding apolypeptide, which polypeptide comprises the amino acid sequence asshown in either SEQ ID NO: 2 or SEQ ID NO: 5, or of a nucleic acidfragment as defined herein, or of a nucleic acid fragment encoding apolypeptide as defined herein. Hybridization probes may be preparedusing the IFN-L sequences provided herein to screen cDNA, genomic orsynthetic DNA libraries for related sequences. Regions of the DNA and/oramino acid sequence of IFN-L polypeptide that exhibit significantidentity to known sequences are readily determined using sequencealignment algorithms as described herein and those regions may be usedto design probes for screening.

[0096] The term “highly stringent conditions” refers to those conditionsthat are designed to permit hybridization of DNA strands whose sequencesare highly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015. M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: APractical Approach Ch. 4 (IRL Press Limited).

[0097] More stringent conditions (such as higher temperature, lowerionic strength, higher formamide, or other denaturing agent) may also beused—however, the rate of hybridization will be affected. Other agentsmay be included in the hybridization and washing buffers for the purposeof reducing non-specific and/or background hybridization. Examples are0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4; however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. See Anderson et al.,Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL PressLimited).

[0098] Factors affecting the stability of DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

T _(m)(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−600/N−0.72(% formamide)

[0099] where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

[0100] The term “moderately stringent conditions” refers to conditionsunder which a DNA duplex with a greater degree of base pair mismatchingthan could occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, “moderately stringent conditions” of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch. It will be appreciated bythose skilled in the art that there is no absolute distinction between“highly stringent conditions” and “moderately stringent conditions.” Forexample, at 0.015 M sodium ion (no formamide), the melting temperatureof perfectly matched long DNA is about 71° C. With a wash at 65° C. (atthe same ionic strength), this would allow for approximately a 6%mismatch. To capture more distantly related sequences, one skilled inthe art can simply lower the temperature or raise the ionic strength.

[0101] A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nt is given by:

Tm=2° C. per A-T base pair+4° C. per G-C base pair

[0102] The sodium ion concentration in 6X salt sodium citrate (SSC) is1M. See Suggs et al., Developmental Biology Using Purified Genes 683(Brown and Fox, eds., 1981).

[0103] High stringency washing conditions for oligonucleotides areusually at a temperature of 0-5° C. below the Tm of the oligonucleotidein 6X SSC, 0.1% SDS.

[0104] In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is at least about 70 percentidentical to the nucleotide sequence as shown in either SEQ ID NO: 1 orSEQ ID NO: 4, or comprise or consist essentially of a nucleotidesequence encoding a polypeptide that is at least about 70 percentidentical to the polypeptide as set forth in either SEQ ID NO: 2 or SEQID NO: 5. In preferred embodiments, the nucleotide sequences are about75 percent, or about 80 percent, or about 85 percent, or about 90percent, or about 95, 96, 97, 98, or 99 percent identical to thenucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, orthe nucleotide sequences encode a polypeptide that is about 75 percent,or about 80 percent, or about 85 percent, or about 90 percent, or about95, 96, 97, 98, or 99 percent identical to the polypeptide sequence asset forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Related nucleic acidmolecules encode polypeptides possessing at least one activity of thepolypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0105] Differences in the nucleic acid sequence may result inconservative and/or non-conservative modifications of the amino acidsequence relative to the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5.

[0106] Conservative modifications to the amino acid sequence of eitherSEQ ID NO: 2 or SEQ ID NO: 5 (and the corresponding modifications to theencoding nucleotides) will produce a polypeptide having functional andchemical characteristics similar to those of IFN-L polypeptides. Incontrast, substantial modifications in the functional and/or chemicalcharacteristics of IFN-L polypeptides may be accomplished by selectingsubstitutions in the amino acid sequence of either SEQ ID NO: 2 or SEQID NO: 5 that differ significantly in their effect on maintaining (a)the structure of the molecular backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

[0107] For example, a “conservative amino acid substitution” may involvea substitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

[0108] Conservative amino acid substitutions also encompassnon-naturally occurring amino acid residues that are typicallyincorporated by chemical peptide synthesis rather than by synthesis inbiological systems. These include peptidomimetics, and other reversed orinverted forms of amino acid moieties.

[0109] Naturally occurring residues may be divided into classes based oncommon side chain properties:

[0110] 1) hydrophobic:. norleucine, Met, Ala, Val, Leu, Ile;

[0111] 2) neutral hydrophilic: Cys, Ser, Thr;

[0112] 3) acidic: Asp, Glu;

[0113] 4) basic: Asn, Gln, His, Lys, Arg;

[0114] 5) residues that influence chain orientation: Gly, Pro; and

[0115] 6) aromatic: Trp, Tyr, Phe.

[0116] For example, non-conservative substitutions may involve theexchange of a member of one of these classes for a member from anotherclass. Such substituted residues may be introduced into regions of thehuman IFN-L polypeptide that are homologous with non-human IFN-Lpolypeptides, or into the non-homologous regions of the molecule.

[0117] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0118] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0119] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

[0120] The following hydrophilicity values have been assigned to theseamino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5 +1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); and tryptophan (−3.4). In making changes basedupon similar hydrophilicity values, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

[0121] Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the IFN-Lpolypeptide, or to increase or decrease the affinity of the IFN-Lpolypeptides described herein. Exemplary amino acid substitutions areset forth in Table I. TABLE I Amino Acid Substitutions Original ResiduesExemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile ValArg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln AsnAsn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met,Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe,Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, CysThr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile,Met, Leu, Phe, Leu Ala, Norleucine

[0122] A skilled artisan will be able to determine suitable variants ofthe polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5using well-known techniques. For identifying suitable areas of themolecule that may be changed without destroying biological activity, oneskilled in the art may target areas not believed to be important foractivity. For example, when similar polypeptides with similar activitiesfrom the same species or from other species are known, one skilled inthe art may compare the amino acid sequence of an IFN-L polypeptide tosuch similar polypeptides. With such a comparison, one can identifyresidues and portions of the molecules that are conserved among similarpolypeptides. It will be appreciated that changes in areas of the IFN-Lmolecule that are not conserved relative to such similar polypeptideswould be less likely to adversely affect the biological activity and/orstructure of an IFN-L polypeptide. One skilled in the art would alsoknow that, even in relatively conserved regions, one may substitutechemically similar amino acids for the naturally occurring residueswhile retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

[0123] Additionally, one skilled in the art can reviewstructure-function studies identifying residues in similar polypeptidesthat are important for activity or structure. In view of such acomparison, one can predict the importance of amino acid residues in anIFN-L polypeptide that correspond to amino acid residues that areimportant for activity or structure in similar polypeptides. One skilledin the art may opt for chemically similar amino acid substitutions forsuch predicted important amino acid residues of IFN-L polypeptides.

[0124] One skilled in the art can also analyze the three-dimensionalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of such information, one skilled in theart may predict the alignment of amino acid residues of IFN-Lpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each amino acid residue. The variants couldbe screened using activity assays known to those with skill in the art.Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change would be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

[0125] A number of scientific publications have been devoted to theprediction of secondary structure. See Moult, 1996, Curr. Opin.Biotechnol. 7:422-27; Chou et al., 1974, Biochemistry 13:222-45; Chou etal., 1974, Biochemistry 113:211-22; Chou et al., 1978, Adv. EnzymolRelat. Areas Mol. Biol. 47:45-48; Chou et al., 1978, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J 26:367-84. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinswhich have a sequence identity of greater than 30%, or similaritygreater than 40%, often have similar structural topologies. The recentgrowth of the protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within the structure of a polypeptide or protein. See Holm et al.,1999, Nucleic Acids Res. 27:244-47. It has been suggested that there area limited number of folds in a given polypeptide or protein and thatonce a critical number of structures have been resolved, structuralprediction will become dramatically more accurate (Brenner et al., 1997,Curr. Opin. Struct. Biol. 7:369-76).

[0126] Additional methods of predicting secondary structure include“threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl etal., 1996, Structure 4:15-19), “profile analysis” (Bowie et al., 1991,Science, 253:164-70; Gribskov et al., 1990, Methods Enzymol. 183:146-59;Gribskov et al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and“evolutionary linkage” (See Holm et al., supra, and Brenner et al.,supra).

[0127] Preferred IFN-L polypeptide variants include glycosylationvariants wherein the number and/or type of glycosylation sites have beenaltered compared to the amino acid sequence set forth in either SEQ IDNO: 2 or SEQ ID NO: 5. In one embodiment, IFN-L polypeptide variantscomprise a greater or a lesser number of N-linked glycosylation sitesthan the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ IDNO: 5. An N-linked glycosylation site is characterized by the sequence:Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as Xmay be any amino acid residue except proline. The substitution of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions that eliminate this sequence will remove an existingN-linked carbohydrate chain. Also provided is a rearrangement ofN-linked carbohydrate chains wherein one or more N-linked glycosylationsites (typically those that are naturally occurring) are eliminated andone or more new N-linked sites are created. Additional preferred IFN-Lvariants include cysteine variants, wherein one or more cysteineresidues are deleted or substituted with another amino acid (e.g.,serine) as compared to the amino acid sequence set forth in either SEQID NO: 2 or SEQ ID NO: 5. Cysteine variants are useful when IFN-Lpolypeptides must be refolded into a biologically active conformationsuch as after the isolation of insoluble inclusion bodies. Cysteinevariants generally have fewer cysteine residues than the native protein,and typically have an even number to minimize interactions resultingfrom unpaired cysteines.

[0128] In other embodiments, related nucleic acid molecules comprise orconsist of a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acidinsertion and wherein the polypeptide has an activity of the polypeptideset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or a nucleotidesequence encoding a polypeptide as set forth in either SEQ ID NO: 2 orSEQ ID NO: 5 with at least one amino acid deletion and wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5. Related nucleic acid molecules also compriseor consist of a nucleotide sequence encoding a polypeptide as set forthin either SEQ ID NO: 2 or SEQ ID NO: 5 wherein the polypeptide has acarboxyl- and/or amino-terminal truncation and further wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5. Related nucleic acid molecules also compriseor consist of a nucleotide sequence encoding a polypeptide as set forthin either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modificationselected from the group consisting of amino acid substitutions, aminoacid insertions, amino acid deletions, carboxyl-terminal truncations,and amino-terminal truncations and wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO: 5.

[0129] In addition, the polypeptide comprising the amino acid sequenceof either SEQ ID NO: 2 or SEQ ID NO: 5, or other IFN-L polypeptide, maybe fused to a homologous polypeptide to form a homodimer or to aheterologous polypeptide to form a heterodimer. Heterologous peptidesand polypeptides include, but are not limited to: an epitope to allowfor the detection and/or isolation of an IFN-L fusion polypeptide; atransmembrane receptor protein or a portion thereof, such as anextracellular domain or a transmembrane and intracellular domain; aligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, orother IFN-L polypeptide.

[0130] Fusions can be made either at the amino-terminus or at thecarboxyl-terminus of the polypeptide comprising the amino acid sequenceset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or other IFN-Lpolypeptide. Fusions may be direct with no linker or adapter molecule ormay be through a linker or adapter molecule. A linker or adaptermolecule may be one or more amino acid residues, typically from about 20to about 50 amino acid residues. A linker or adapter molecule may alsobe designed with a cleavage site for a DNA restriction endonuclease orfor a protease to allow for the separation of the fused moieties. Itwill be appreciated that once constructed, the fusion polypeptides canbe derivatized according to the methods described herein.

[0131] In a further embodiment of the invention, the polypeptidecomprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO:5, or other IFN-L polypeptide, is fused to one or more domains of an Fcregion of human IgG. Antibodies comprise two functionally independentparts, a variable domain known as “Fab,” that binds an antigen, and aconstant domain known as “Fc,” that is involved in effector functionssuch as complement activation and attack by phagocytic cells. An Fc hasa long serum half-life, whereas an Fab is short-lived. Capon et al.,1989, Nature 337:525-31. When constructed together with a therapeuticprotein, an Fc domain can provide longer half-life or incorporate suchfunctions as Fc receptor binding, protein A binding, complementfixation, and perhaps even placental transfer. Id. Table II summarizesthe use of certain Fc fusions known in the art. TABLE II Fc Fusion withTherapeutic Proteins Form of Fc Fusion partner Therapeutic implicationsReference IgG1 N-terminus of Hodgkin's disease; U.S. Pat. No. CD30-Lanaplastic lymphoma; T- 5,480,981 cell leukemia Murine Fcγ2a IL-10anti-inflammatory; Zheng et al., 1995, J transplant rejection Immunol.154:5590-600 IgG1 TNF receptor septic shock Fisher et al., 1996, N.Engl; J. Med. 334:1697- 1702; Van Zee et al., 1996, J. Immunol.156:2221-30 IgG, IgA, IgM, TNF receptor inflammation, U.S. Pat. No. orIgE autoimmune disorders 5,808,029 (excluding the first domain) IgG1 CD4receptor AIDS Capon et al., 1989, Nature 337: 525-31 IgG1, N-terminusanti-cancer, antiviral Harvill et al., 1995, IgG3 of IL-2 Immunotech.1:95-105 IgG1 C-terminus of osteoarthritis; WO 97/23614 OPG bone densityIgG1 N-terminus of anti-obesity PCT/US 97/23183, filed leptin December11, 1997 Human Ig Cγ1 CTLA-4 autoimmune disorders Linsley, 1991, J. Exp.Med., 174:561-69

[0132] In one example, a human IgG hinge, CH2, and CH3 region may befused at either the amino-terminus or carboxyl-terminus of the IFN-Lpolypeptides using methods known to the skilled artisan. In anotherexample, a human IgG hinge, CH2, and CH3 region may be fused at eitherthe amino-terminus or carboxyl-terminus of an IFN-L polypeptide fragment(e.g., the predicted extracellular portion of IFN-L polypeptide).

[0133] The resulting IFN-L fusion polypeptide may be purified by use ofa Protein A affinity column. Peptides and proteins fused to an Fc regionhave been found to exhibit a substantially greater half-life in vivothan the unfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time, orreduced aggregation.

[0134] Identity and similarity of related nucleic acid molecules andpolypeptides are readily calculated by known methods. Such methodsinclude, but are not limited to those described in ComputationalMolecular Biology (A. M. Lesk, ed., Oxford University Press 1988);Biocomputing. Informatics and Genome Projects (D. W. Smith, ed.,Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A. M.Griffin and H. G. Griffin, eds., Humana Press 1994); G. von Heinle,Sequence Analysis in Molecular Biology (Academic Press 1987); SequenceAnalysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press1991); and Carillo et al., 1988, SIAM J. Applied Math., 48:1073.

[0135] Preferred methods to determine identity and/or similarity aredesigned to give the largest match between the sequences tested. Methodsto determine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,1990, J. Mol. Biol. 215:403-10). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (Altschul et al, BLAST Manual (NCB NLM NIH, Bethesda,Md.); Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

[0136] Certain alignment schemes for aligning two amino acid sequencesmay result in the matching of only a short region of the two sequences,and this small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the claimed is polypeptide.

[0137] For example, using the computer algorithm GAP (Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), two polypeptides forwhich the percent sequence identity is to be determined are aligned foroptimal matching of their respective amino acids (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3X the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 0.1X the gap opening penalty), as well as a comparison matrixsuch as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.A standard comparison matrix is also used by the algorithm (see Dayhoffet al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)(PAM250comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci USA89:10915-19 (BLOSUM 62 comparison matrix)).

[0138] Preferred parameters for polypeptide sequence comparison includethe following:

[0139] Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-53;

[0140] Comparison matrix: BLOSUM 62 (Henikoffet al., supra);

[0141] Gap Penalty: 12

[0142] Gap Length Penalty: 4

[0143] Threshold of Similarity: 0

[0144] The GAP program is useful with the above parameters. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps) using the GAPalgorithm.

[0145] Preferred parameters for nucleic acid molecule sequencecomparison include the following:

[0146] Algorithm: Needleman and Wunsch, supra;

[0147] Comparison matrix: matches=+10, mismatch=0

[0148] Gap Penalty: 50

[0149] Gap Length Penalty: 3

[0150] The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

[0151] Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, and thresholds of similarity may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

[0152] Nucleic Acid Molecules

[0153] The nucleic acid molecules encoding a polypeptide comprising theamino acid sequence of an IFN-L polypeptide can readily be obtained in avariety of ways including, without limitation, chemical synthesis, cDNAor genomic library screening, expression library screening, and/or PCRamplification of cDNA.

[0154] Recombinant DNA methods used herein are generally those set forthin Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994). The invention provides for nucleic acid molecules as describedherein and methods for obtaining such molecules.

[0155] Where a gene encoding the amino acid sequence of an IFN-Lpolypeptide has been identified from one species, all or a portion ofthat gene may be used as a probe to identify orthologs or related genesfrom the same species. The probes or primers may be used to screen cDNAlibraries from various tissue sources believed to express the IFN-Lpolypeptide. In addition, part or all of a nucleic acid molecule havingthe sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4 may beused to screen a genomic library to identify and isolate a gene encodingthe amino acid sequence of an IFN-L polypeptide. Typically, conditionsof moderate or high stringency will be employed for screening tominimize the number of false positives obtained from the screening.

[0156] Nucleic acid molecules encoding the amino acid sequence of IFN-Lpolypeptides may also be identified by expression cloning which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingan antibody or other binding partner (e.g., receptor or ligand) tocloned proteins that are expressed and displayed on a host cell surface.The antibody or binding partner is modified with a detectable label toidentify those cells expressing the desired clone.

[0157] Recombinant expression techniques conducted in accordance withthe descriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence that encodes the amino acid sequenceof an IFN-L polypeptide into an appropriate vector, one skilled in theart can readily produce large quantities of the desired nucleotidesequence. The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of an IFN-L polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded IFN-L polypeptide may be produced in largeamounts.

[0158] Another method for obtaining a suitable nucleic acid sequence isthe polymerase chain reaction (PCR). In this method, cDNA is preparedfrom poly(A)+RNA or total RNA using the enzyme reverse transcriptase.Two primers, typically complementary to two separate regions of cDNAencoding the amino acid sequence of an IFN-L polypeptide, are then addedto the cDNA along with a polymerase such as Taq polymerase, and thepolymerase amplifies the cDNA region between the two primers.

[0159] Another means of preparing a nucleic acid molecule encoding theamino acid sequence of an IFN-L polypeptide is chemical synthesis usingmethods well known to the skilled artisan such as those described byEngels et al., 1989, Angew. Chem. Intl. Ed. 28:716-34. These methodsinclude, inter alia, the phosphotriester, phosphoramidite, andH-phosphonate methods for nucleic acid synthesis. A preferred method forsuch chemical synthesis is polymer-supported synthesis using standardphosphoramidite chemistry. Typically, the DNA encoding the amino acidsequence of an IFN-L polypeptide will be several hundred nucleotides inlength. Nucleic acids larger than about 100 nucleotides can besynthesized as several fragments using these methods. The fragments canthen be ligated together to form the full-length nucleotide sequence ofan IFN-L gene. Usually, the DNA fragment encoding the amino-terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of theIFN-L polypeptide, depending on whether the polypeptide produced in thehost cell is designed to be secreted from that cell. Other methods knownto the skilled artisan may be used as well.

[0160] In certain embodiments, nucleic acid variants contain codonswhich have been altered for optimal expression of an IFN-L polypeptidein a given host cell. Particular codon alterations will depend upon theIFN-L polypeptide and host cell selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Eco_high.Cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0 (Genetics Computer Group, Madison,Wis.). Other useful codon frequency tables include “Celegans_high.cod,”“Celegans_low.cod,” “Drosophila_high.cod,” “Human_high.cod,”“Maize_high.cod,” and “Yeast_high.cod.”

[0161] In some cases, it may be desirable to prepare nucleic acidmolecules encoding IFN-L polypeptide variants. Nucleic acid moleculesencoding variants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

[0162] Vectors and Host Cells

[0163] A nucleic acid molecule encoding the amino acid sequence of anIFN-L polypeptide is inserted into an appropriate expression vectorusing standard ligation techniques. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of an IFN-L polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether an IFN-L polypeptide is to be post-translationallymodified (e.g., glycosylated and/or phosphorylated). If so, yeast,insect, or mammalian host cells are preferable. For a review ofexpression vectors, see Meth. Enz., vol. 185 (D. V. Goeddel, ed.,Academic Press 1990).

[0164] Typically, expression vectors used in any of the host cells willcontain sequences for plasmid maintenance and for cloning and expressionof exogenous nucleotide sequences. Such sequences, collectively referredto as “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

[0165] Optionally, the vector may contain a “tag”-encoding sequence,i.e., an oligonucleotide molecule located at the 5′ or 3′ end of theIFN-L polypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the IFN-L polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified IFN-L polypeptide byvarious means such as using certain peptidases for cleavage.

[0166] Flanking sequences may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof flanking sequences from more than one source), or synthetic, or theflanking sequences may be native sequences which normally function toregulate IFN-L polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

[0167] Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein—other than the IFN-L gene flankingsequences—will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

[0168] Where all or only a portion of the flanking sequence is known, itmay be obtained using PCR and/or by screening a genomic library with asuitable oligonucleotide and/or flanking sequence fragment from the sameor another species. Where the flanking sequence is not known, a fragmentof DNA containing a flanking sequence may be isolated from a largerpiece of DNA that may contain, for example, a coding sequence or evenanother gene or genes. Isolation may be accomplished by restrictionendonuclease digestion to produce the proper DNA fragment followed byisolation using agarose gel purification, Qiagen® column chromatography(Chatsworth, Calif.), or other methods known to the skilled artisan. Theselection of suitable enzymes to accomplish this purpose will be readilyapparent to one of ordinary skill in the art.

[0169] An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of an IFN-L polypeptide. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector.

[0170] For example, the origin of replication from the plasmid pBR322(New England Biolabs, Beverly, Mass.) is suitable for most gram-negativebacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it contains theearly promoter).

[0171] A transcription termination sequence is typically located 3′ ofthe end of a polypeptide coding region and serves to terminatetranscription. Usually, a transcription termination sequence inprokaryotic cells is a G−C rich fragment followed by a poly-T sequence.While the sequence is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing methods for nucleic acid synthesis such as those described herein.

[0172] A selectable marker gene element encodes a protein necessary forthe survival and growth of a host cell grown in a selective culturemedium. Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

[0173] Other selection genes may be used to amplify the gene that willbe expressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whereinonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes an IFN-L polypeptide. As a result, increased quantitiesof IFN-L polypeptide are synthesized from the amplified DNA.

[0174] A ribosome binding site is usually necessary for translationinitiation of mRNA and is characterized by a Shine-Dalgamo sequence(prokaryotes) or a Kozak sequence (eukaryotes). The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of an IFN-Lpolypeptide to be expressed. The Shine-Dalgarno sequence is varied butis typically a polypurine (i.e., having a high A−G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth herein and used in aprokaryotic vector.

[0175] A leader, or signal, sequence may be used to direct an IFN-Lpolypeptide out of the host cell. Typically, a nucleotide sequenceencoding the signal sequence is positioned in the coding region of anIFN-L nucleic acid molecule, or directly at the 5′ end of an IFN-Lpolypeptide coding region. Many signal sequences have been identified,and any of those that are functional in the selected host cell may beused in conjunction with an IFN-L nucleic acid molecule. Therefore, asignal sequence may be homologous (naturally occurring) or heterologousto the IFN-L nucleic acid molecule. Additionally, a signal sequence maybe chemically synthesized using methods described herein. In most cases,the secretion of an IFN-L polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the signalpeptide from the secreted IFN-L polypeptide. The signal sequence may bea component of the vector, or it may be a part of an IFN-L nucleic acidmolecule that is inserted into the vector.

[0176] Included within the scope of this invention is the use of eithera nucleotide sequence encoding a native IFN-L polypeptide signalsequence joined to an IFN-L polypeptide coding region or a nucleotidesequence encoding a heterologous signal sequence joined to an IFN-Lpolypeptide coding region. The heterologous signal sequence selectedshould be one that is recognized and processed, i.e., cleaved by asignal peptidase, by the host cell. For prokaryotic host cells that donot recognize and process the native IFN-L polypeptide signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, or heat-stable enterotoxin II leaders. For yeastsecretion, the native IFN-L polypeptide signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

[0177] In some cases, such as where glycosylation is desired in aeukaryotic host cell expression system, one may manipulate the variouspresequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired IFN-L polypeptide, if the enzymecuts at such area within the mature polypeptide.

[0178] In many cases, transcription of a nucleic acid molecule isincreased by the presence of one or more introns in the vector; this isparticularly true where a polypeptide is produced in eukaryotic hostcells, especially mammalian host cells. The introns used may benaturally occurring within the IFN-L gene especially where the gene usedis a full-length genomic sequence or a fragment thereof Where the intronis not naturally occurring within the gene (as for most cDNAs), theintron may be obtained from another source. The position of the intronwith respect to flanking sequences and the IFN-L gene is generallyimportant, as the intron must be transcribed to be effective. Thus, whenan IFN-L cDNA molecule is being transcribed, the preferred position forthe intron is 3′ to the transcription start site and 5′ to the poly-Atranscription termination sequence. Preferably, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the coding sequence. Any intron from anysource, including viral, prokaryotic and eukaryotic (plant or animal)organisms, may be used to practice this invention, provided that it iscompatible with the host cell into which it is inserted. Also includedherein are synthetic introns. Optionally, more than one intron may beused in the vector.

[0179] The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the IFN-L polypeptide.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control the transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding IFN-L polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native IFN-L promoter sequence may be used to direct amplificationand/or expression of an IFN-L nucleic acid molecule. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter, and if it is compatible with the host cell system that hasbeen selected for use.

[0180] Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase; atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any useful restriction sites.

[0181] Suitable promoters for use with yeast hosts are also well knownin the art. Yeast enhancers are advantageously used with yeastpromoters. Suitable promoters for use with mammalian host cells are wellknown and include, but are not limited to, those obtained from thegenomes of viruses such as polyoma virus, fowlpox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

[0182] Additional promoters which may be of interest in controllingIFN-L gene expression include, but are not limited to: the SV40 earlypromoter region (Bemoist and Chambon, 1981, Nature 290:304-10); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987,Hepatology 7:425-515); the insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); themouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95);the albumin gene control region which is active in liver (Pinkert etal., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein genecontrol region which is active in liver (Krumlauf et al., 1985, Mol.Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); thealpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-78).

[0183] An enhancer sequence may be inserted into the vector to increasethe transcription of a DNA encoding an IFN-L polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to an IFN-L nucleic acidmolecule, it is typically located at a site 5′ from the promoter.

[0184] Expression vectors of the invention may be constructed from astarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the flanking sequences described herein are not alreadypresent in the vector, they may be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

[0185] Preferred vectors for practicing this invention are those whichare compatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha(PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island,N.Y.).

[0186] Additional suitable vectors include, but are not limited to,cosmids, plasmids, or modified viruses, but it will be appreciated thatthe vector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems, La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeastor virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.).

[0187] After the vector has been constructed and a nucleic acid moleculeencoding an IFN-L polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an IFN-L polypeptide into a selected hostcell may be accomplished by well known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

[0188] Host cells may be prokaryotic host cells (such as E. coli) oreukaryotic host cells (such as a yeast, insect, or vertebrate cell). Thehost cell, when cultured under appropriate conditions, synthesizes anIFN-L polypeptide which can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

[0189] A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include, but are not limited to, mammalian cells, such asChinese hamster ovary cells (CHO), CHO DHFR(−) cells (Urlaub et al.,1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney(HEK) 293 or 293T cells, or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening, product production, and purification are knownin the art. Other suitable mammalian cell lines, are the monkey COS-1and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines. Each of these cell lines is known by and available to thoseskilled in the art of protein expression.

[0190] Similarly useful as host cells suitable for the present inventionare bacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

[0191] Many strains of yeast cells known to those skilled in the art arealso available as host cells for the expression of the polypeptides ofthe present invention. Preferred yeast cells include, for example,Saccharomyces cerivisae and Pichia pastoris.

[0192] Additionally, where desired, insect cell systems may be utilizedin the methods of the present invention. Such systems are described, forexample, in Kitts et al., 1993, Biotechniques, 14:810-17; Lucklow, 1993,Curr. Opin. Biotechnol. 4:564-72; and Lucklow et al., 1993, J. Virol.,67:4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

[0193] One may also use transgenic animals to express glycosylated IFN-Lpolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce IFN-Lpolypeptides, however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

[0194] Polypeptide Production

[0195] Host cells comprising an IFN-L polypeptide expression vector maybe cultured using standard media well known to the skilled artisan. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cellsinclude, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells include Roswell ParkMemorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium(MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which maybe supplemented with serum and/or growth factors as necessary for theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

[0196] Typically, an antibiotic or other compound useful for selectivegrowth of transfected or transformed cells is added as a supplement tothe media. The compound to be used will be dictated by the selectablemarker element present on the plasmid with which the host cell wastransformed. For example, where the selectable marker element iskanamycin resistance, the compound added to the culture medium will bekanamycin. Other compounds for selective growth include ampicillin,tetracycline, and neomycin.

[0197] The amount of an IFN-L polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, High PerformanceLiquid Chromatography (HPLC) separation, immunoprecipitation, and/oractivity assays such as DNA binding gel shift assays.

[0198] If an IFN-L polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. If however, the IFN-L polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram-negative bacteriahost cells).

[0199] For an IFN-L polypeptide situated in the host cell cytoplasmand/or nucleus (for eukaryotic host cells) or in the cytosol (forbacterial host cells), the intracellular material (including inclusionbodies for gram-negative bacteria) can be extracted from the host cellusing any standard technique known to the skilled artisan. For example,the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

[0200] If an IFN-L polypeptide has formed inclusion bodies in thecytosol, the inclusion bodies can often bind to the inner and/or outercellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with a chaotropic agent such as a detergent,guanidine, guanidine derivatives, urea, or urea derivatives in thepresence of a reducing agent such as dithiothreitol at alkaline pH ortris carboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The solubilized IFN-L polypeptide canthen be analyzed using gel electrophoresis, immunoprecipitation, or thelike. If it is desired to isolate the IFN-L polypeptide, isolation maybe accomplished using standard methods such as those described hereinand in Marston et al., 1990, Meth. Enz., 182:264-75.

[0201] In some cases, an IFN-L polypeptide may not be biologicallyactive upon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkagescan be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridges. Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolventmay be used or may be needed to increase the efficiency of therefolding, and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, arginine andthe like.

[0202] If inclusion bodies are not formed to a significant degree uponexpression of an IFN-L polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide may be further isolated from the supernatantusing methods such as those described herein.

[0203] The purification of an IFN-L polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (IFN-Lpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl- or amino-terminus, it may be purified in a one-stepprocess by passing the solution through an affinity column where thecolumn matrix has a high affinity for the tag.

[0204] For example, polyhistidine binds with great affinity andspecificity to nickel. Thus, an affinity column of nickel (such as theQiagen® nickel columns) can be used for purification of IFN-Lpolypeptide/polyHis. See, e.g. Current Protocols in Molecular Biology §10.11.8 (Ausubel et al, eds., Green Publishers Inc. and Wiley and Sons1993).

[0205] Additionally, IFN-L polypeptides may be purified through the useof a monoclonal antibody that is capable of specifically recognizing andbinding to an IFN-L polypeptide.

[0206] Other suitable procedures for purification include, withoutlimitation, affinity chromatography, immunoaffinity chromatography, ionexchange chromatography, molecular sieve chromatography, HPLC,electrophoresis (including native gel electrophoresis) followed by gelelution, and preparative isoelectric focusing (“Isoprime”machine/technique, Hoefer Scientific, San Francisco, Calif.). In somecases, two or more purification techniques may be combined to achieveincreased purity.

[0207] IFN-L polypeptides may also be prepared by chemical synthesismethods (such as solid phase peptide synthesis) using techniques knownin the art such as those set forth by Merrifield et al., 1963, J. Am.Chem. Soc. 85:2149; Houghten et al., 1985, Proc Natl Acad. Sci. USA82:5132; and Stewart and Young, Solid Phase Peptide Synthesis (PierceChemical Co. 1984). Such polypeptides may be synthesized with or withouta methionine on the amino-terminus. Chemically synthesized IFN-Lpolypeptides may be oxidized using methods set forth in these referencesto form disulfide bridges. Chemically synthesized IFN-L polypeptides areexpected to have comparable biological activity to the correspondingIFN-L polypeptides produced recombinantly or purified from naturalsources, and thus may be used interchangeably with a recombinant ornatural IFN-L polypeptide.

[0208] Another means of obtaining IFN-L polypeptide is via purificationfrom biological samples such as source tissues and/or fluids in whichthe IFN-L polypeptide is naturally found. Such purification can beconducted using methods for protein purification as described herein.The presence of the IFN-L polypeptide during purification may bemonitored, for example, using an antibody prepared against recombinantlyproduced IFN-L polypeptide or peptide fragments thereof.

[0209] A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for IFN-L polypeptide. See, e.g.Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:12297-303, whichdescribes the production of fusion proteins between an mRNA and itsencoded peptide. See also, Roberts, 1999, Curr. Opin. Chem. Biol.3:268-73. Additionally, U.S. Pat. No. 5,824,469 describes methods forobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those that exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

[0210] U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483describe processes for producing peptides or polypeptides. This is doneby producing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

[0211] Another method for producing peptides or polypeptides isdescribed in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Knownas “Random Activation of Gene Expression for Gene Discovery” (RAGE-GD),the process involves the activation of endogenous gene expression orover-expression of a gene by in situ recombination methods. For example,expression of an endogenous gene is activated or increased byintegrating a regulatory sequence into the target cell which is capableof activating expression of the gene by non-homologous or illegitimaterecombination. The target DNA is first subjected to radiation, and agenetic promoter inserted. The promoter eventually locates a break atthe front of a gene, initiating transcription of the gene. This resultsin expression of the desired peptide or polypeptide.

[0212] It will be appreciated that these methods can also be used tocreate comprehensive IFN-L polypeptide expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

[0213] Synthesis

[0214] It will be appreciated by those skilled in the art that thenucleic acid and polypeptide molecules described herein may be producedby recombinant and other means.

[0215] Selective Binding Agents

[0216] The term “selective binding agent” refers to a molecule that hasspecificity for one or more IFN-L polypeptides. Suitable selectivebinding agents include, but are not limited to, antibodies andderivatives thereof, polypeptides, and small molecules. Suitableselective binding agents may be prepared using methods known in the art.An exemplary IFN-L polypeptide selective binding agent of the presentinvention is capable of binding a certain portion of the IFN-Lpolypeptide thereby inhibiting the binding of the polypeptide to anIFN-L polypeptide receptor.

[0217] Selective binding agents such as antibodies and antibodyfragments that bind IFN-L polypeptides are within the scope of thepresent invention. The antibodies may be polyclonal includingmonospecific polyclonal; monoclonal (MAbs); recombinant; chimeric;humanized, such as CDR-grafted; human; single chain; and/or bispecific;as well as fragments; variants; or derivatives thereof. Antibodyfragments include those portions of the antibody that bind to an epitopeon the IFN-L polypeptide. Examples of such fragments include Fab andF(ab′) fragments generated by enzymatic cleavage of full-lengthantibodies. Other binding fragments include those generated byrecombinant DNA techniques, such as the expression of recombinantplasmids containing nucleic acid sequences encoding antibody variableregions.

[0218] Polyclonal antibodies directed toward an IFN-L polypeptidegenerally are produced in animals (e.g., rabbits or mice) by means ofmultiple subcutaneous or intraperitoneal injections of IFN-L polypeptideand an adjuvant. It may be useful to conjugate an IFN-L polypeptide to acarrier protein that is immunogenic in the species to be immunized, suchas keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-IFN-L antibody titer.

[0219] Monoclonal antibodies directed toward IFN-L polypeptides areproduced using any method that provides for the production of antibodymolecules by continuous cell lines in culture. Examples of suitablemethods for preparing monoclonal antibodies include the hybridomamethods of Kohler et al., 1975, Nature 256:495-97 and the human B-cellhybridoma method (Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al.,Monoclonal Antibody Production Techniques and Applications 51-63 (MarcelDekker, Inc., 1987). Also provided by the invention are hybridoma celllines that produce monoclonal antibodies reactive with IFN-Lpolypeptides.

[0220] Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy (H) and/or light (L) chain is identical with or homologousto a corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985,Proc. Natl. Acad. Sci. 81:6851-55.

[0221] In another embodiment, a monoclonal antibody of the invention isa “humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art (Jones etal., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27;Verhoeyen et al., 1988, Science 239:1534-36), by substituting at least aportion of a rodent complementarity-determining region (CDR) for thecorresponding regions of a human antibody.

[0222] Also encompassed by the invention are human antibodies that bindIFN-L polypeptides. Using transgenic animals (e.g., mice) that arecapable of producing a repertoire of human antibodies in the absence ofendogenous immunoglobulin production such antibodies are produced byimmunization with an IFN-L polypeptide antigen (i.e., having at least 6contiguous amino acids), optionally conjugated to a carrier. See, e.g.,Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovitset al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year inImmuno. 7:33. In one method, such transgenic animals are produced byincapacitating the endogenous loci encoding the heavy and lightimmunoglobulin chains therein, and inserting loci encoding human heavyand light chain proteins into the genome thereof. Partially modifiedanimals, that is those having less than the full complement ofmodifications, are then cross-bred to obtain an animal having all of thedesired immune system modifications. When administered an immunogen,these transgenic animals produce antibodies with human (rather than,e.g., murine) amino acid sequences, including variable regions which areimmunospecific for these antigens. See PCT App. Nos. PCT/US96/05928 andPCT/US93/06926. Additional methods are described in U.S. Pat. No.5,545,807, PCT application Ser. Nos. PCT/US91/245 and PCT/GB89/01207,and in European Patent Nos. 546073B1 and 546073A1. Human antibodies canalso be produced by the expression of recombinant DNA in host cells orby expression in hybridoma cells as described herein.

[0223] In an alternative embodiment, human antibodies can also beproduced from phage-display libraries (Hoogenboom et al., 1991, J Mol.Biol. 227:381; Marks et al., 1991, J Mol. Biol. 222:581). Theseprocesses mimic immune selection through the display of antibodyrepertoires on the surface of filamentous bacteriophage, and subsequentselection of phage by their binding to an antigen of choice. One suchtechnique is described in PCT application Ser. No. PCT/US98/17364, whichdescribes the isolation of high affinity and functional agonisticantibodies for MPL- and msk- receptors using such an approach.

[0224] Chimeric, CDR grafted, and humanized antibodies are typicallyproduced by recombinant methods. Nucleic acids encoding the antibodiesare introduced into host cells and expressed using materials andprocedures described herein. In a preferred embodiment, the antibodiesare produced in mammalian host cells, such as CHO cells. Monoclonal(e.g., human) antibodies may be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

[0225] The anti-IFN-L antibodies of the invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press, Inc.,1987)) for the detection and quantitation of IFN-L polypeptides. Theantibodies will bind IFN-L polypeptides with an affinity that isappropriate for the assay method being employed.

[0226] For diagnostic applications, in certain embodiments, anti-IFN-Lantibodies may be labeled with a detectable moiety. The detectablemoiety can be any one that is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc, ¹¹¹In, or⁶⁷Ga; a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodantine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase (Bayer, et al.,1990, Meth. Enz. 184:138-63).

[0227] Competitive binding assays rely on the ability of a labeledstandard (e.g., an IFN-L polypeptide, or an immunologically reactiveportion thereof) to compete with the test sample analyte (an IFN-Lpolypeptide) for binding with a limited amount of anti-IFN-L antibody.The amount of an IFN-L polypeptide in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0228] Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

[0229] The selective binding agents, including anti-IFN-L antibodies,are also useful for in vivo imaging. An antibody labeled with adetectable moiety may be administered to an animal, preferably into thebloodstream, and the presence and location of the labeled antibody inthe host assayed. The antibody may be labeled with any moiety that isdetectable in an animal, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

[0230] Selective binding agents of the invention, including antibodies,may be used as therapeutics. These therapeutic agents are generallyagonists or antagonists, in that they either enhance or reduce,respectively, at least one of the biological activities of an IFN-Lpolypeptide. In one embodiment, antagonist antibodies of the inventionare antibodies or binding fragments thereof which are capable ofspecifically binding to an IFN-L polypeptide and which are capable ofinhibiting or eliminating the functional activity of an IFN-Lpolypeptide in vivo or in vitro. In preferred embodiments, the selectivebinding agent, e.g. an antagonist antibody, will inhibit the functionalactivity of an IFN-L polypeptide by at least about 50%, and preferablyby at least about 80%. In another embodiment, the selective bindingagent may be an anti-IFN-L polypeptide antibody that is capable ofinteracting with an IFN-L polypeptide binding partner (a ligand orreceptor) thereby inhibiting or eliminating IFN-L polypeptide activityin vitro or in vivo. Selective binding agents, including agonist andantagonist anti-IFN-L polypeptide antibodies, are identified byscreening assays that are well known in the art.

[0231] The invention also relates to a kit comprising IFN-L selectivebinding agents (such as antibodies) and other reagents useful fordetecting IFN-L polypeptide levels in biological samples. Such reagentsmay include a detectable label, blocking serum, positive and negativecontrol samples, and detection reagents.

[0232] Microarrays

[0233] It will be appreciated that DNA microarray technology can beutilized in accordance with the present invention. DNA microarrays areminiature, high-density arrays of nucleic acids positioned on a solidsupport, such as glass. Each cell or element within the array containsnumerous copies of a single nucleic acid species that acts as a targetfor hybridization with a complementary nucleic acid sequence (e.g.,mRNA). In expression profiling using DNA microarray technology, mRNA isfirst extracted from a cell or tissue sample and then convertedenzymatically to fluorescently labeled cDNA. This material is hybridizedto the microarray and unbound cDNA is removed by washing. The expressionof discrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA that is specifically bound toeach target nucleic acid molecule. In this way, the expression ofthousands of genes can be quantitated in a high throughput, parallelmanner from a single sample of biological material.

[0234] This high throughput expression profiling has a broad range ofapplications with respect to the IFN-L molecules of the invention,including, but not limited to: the identification and validation ofIFN-L disease-related genes as targets for therapeutics; moleculartoxicology of related IFN-L molecules and inhibitors thereof;stratification of populations and generation of surrogate markers forclinical trials; and enhancing related IFN-L polypeptide small moleculedrug discovery by aiding in the identification of selective compounds inhigh throughput screens.

[0235] Chemical Derivatives

[0236] Chemically modified derivatives of IFN-L polypeptides may beprepared by one skilled in the art, given the disclosures describedherein. IFN-L polypeptide derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical groups. Thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5, or other IFN-L polypeptide, may be modified by thecovalent attachment of one or more polymers. For example, the polymerselected is typically water-soluble so that the protein to which it isattached does not precipitate in an aqueous environment, such as aphysiological environment. Included within the scope of suitablepolymers is a mixture of polymers. Preferably, for therapeutic use ofthe end-product preparation, the polymer will be pharmaceuticallyacceptable.

[0237] The polymers each may be of any molecular weight and may bebranched or unbranched. The polymers each typically have an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water-soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight).The average molecular weight of each polymer is preferably between about5 kDa and about 50 kDa, more preferably between about 12 kDa and about40 kDa and most preferably between about 20 kDa and about 35 kDa.

[0238] Suitable water-soluble polymers or mixtures thereof include, butare not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, polyethylene glycol (PEG) (including the forms of PEG thathave been used to derivatize proteins, including mono-(C₁-C₁₀), alkoxy-,or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol,dextran (such as low molecular weight dextran of, for example, about 6kD), cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached IFN-L polypeptide multimers.

[0239] In general, chemical derivatization may be performed under anysuitable condition used to react a protein with an activated polymermolecule. Methods for preparing chemical derivatives of polypeptideswill generally comprise the steps of: (a) reacting the polypeptide withthe activated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of either SEQ ID NO: 2 orSEQ ID NO: 5, or other IFN-L polypeptide, becomes attached to one ormore polymer molecules, and (b) obtaining the reaction products. Theoptimal reaction conditions will be determined based on known parametersand the desired result. For example, the larger the ratio of polymermolecules to protein, the greater the percentage of attached polymermolecule. In one embodiment, the IFN-L polypeptide derivative may have asingle polymer molecule moiety at the amino-terminus. See, e.g., U.S.Pat. No. 5,234,784.

[0240] The pegylation of a polypeptide may be specifically carried outusing any of the pegylation reactions known in the art. Such reactionsare described, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and0401384; and U.S. Pat. No. 4,179,337. For example, pegylation may becarried out via an acylation reaction or an alkylation reaction with areactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer) as described herein. For the acylation reactions,a selected polymer should have a single reactive ester group. Forreductive alkylation, a selected polymer should have a single reactivealdehyde group. A reactive aldehyde is, for example, polyethylene glycolpropionaldehyde, which is water stable, or mono C₁-C₁₀ alkoxy or aryloxyderivatives thereof (see U.S. Pat. No. 5,252,714).

[0241] In another embodiment, IFN-L polypeptides may be chemicallycoupled to biotin. The biotin/IFN-L polypeptide molecules are thenallowed to bind to avidin, resulting in tetravalent avidin/biotin/IFN-Lpolypeptide molecules. IFN-L polypeptides may also be covalently coupledto dinitrophenol (DNP) or trinitrophenol (TNP) and the resultingconjugates precipitated with anti-DNP or anti-TNP-IgM to form decamericconjugates with a valency of 10.

[0242] Generally, conditions that may be alleviated or modulated by theadministration of the present IFN-L polypeptide derivatives includethose described herein for IFN-L polypeptides. However, the IFN-Lpolypeptide derivatives disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

[0243] Genetically Engineered Non-Human Animals

[0244] Additionally included within the scope of the present inventionare non-human animals such as mice, rats, or other rodents; rabbits,goats, sheep, or other farm animals, in which the genes encoding nativeIFN-L polypeptide have been disrupted (i.e., “knocked out”) such thatthe level of expression of IFN-L polypeptide is significantly decreasedor completely abolished. Such animals may be prepared using techniquesand methods such as those described in U.S. Pat. No. 5,557,032.

[0245] The present invention further includes non-human animals such asmice, rats, or other rodents; rabbits, goats, sheep, or other farmanimals, in which either the native form of an IFN-L gene for thatanimal or a heterologous IFN-L gene is over-expressed by the animal,thereby creating a “transgenic” animal. Such transgenic animals may beprepared using well known methods such as those described in U.S. Pat.No 5,489,743 and PCT Pub. No. WO 94/28122.

[0246] The present invention further includes non-human animals in whichthe promoter for one or more of the IFN-L polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native IFN-L polypeptides.

[0247] These non-human animals may be used for drug candidate screening.In such screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the IFN-L gene. In certain embodiments, the amount ofIFN-L polypeptide that is produced may be measured after the exposure ofthe animal to the drug candidate. Additionally, in certain embodiments,one may detect the actual impact of the drug candidate on the animal.For example, over-expression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

[0248] Assaying for Other Modulators of IFN-L Polypeptide Activity

[0249] In some situations, it may be desirable to identify moleculesthat are modulators, i.e., agonists or antagonists, of the activity ofIFN-L polypeptide. Natural or synthetic molecules that modulate IFN-Lpolypeptide may be identified using one or more screening assays, suchas those described herein. Such molecules may be administered either inan ex vivo manner or in an in vivo manner by injection, or by oraldelivery, implantation device, or the like.

[0250] “Test molecule” refers to a molecule that is under evaluation forthe ability to modulate (i.e., increase or decrease) the activity of anIFN-L polypeptide. Most commonly, a test molecule will interact directlywith an IFN-L polypeptide. However, it is also contemplated that a testmolecule may also modulate IFN-L polypeptide activity indirectly, suchas by affecting IFN-L gene expression, or by binding to an IFN-Lpolypeptide binding partner (e.g., receptor or ligand). In oneembodiment, a test molecule will bind to an IFN-L polypeptide with anaffinity constant of at least about 10⁻⁶ M, preferably about 10⁻⁸ M,more preferably about 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

[0251] Methods for identifying compounds that interact with IFN-Lpolypeptides are encompassed by the present invention. In certainembodiments, an IFN-L polypeptide is incubated with a test moleculeunder conditions that permit the interaction of the test molecule withan IFN-L polypeptide, and the extent of the interaction is measured. Thetest molecule can be screened in a substantially purified form or in acrude mixture.

[0252] In certain embodiments, an IFN-L polypeptide agonist orantagonist may be a protein, peptide, carbohydrate, lipid, or smallmolecular weight molecule that interacts with IFN-L polypeptide toregulate its activity. Molecules which regulate IFN-L polypeptideexpression include nucleic acids which are complementary to nucleicacids encoding an IFN-L polypeptide, or are complementary to nucleicacids sequences which direct or control the expression of IFN-Lpolypeptide, and which act as anti-sense regulators of expression.

[0253] Once a test molecule has been identified as interacting with anIFN-L polypeptide, the molecule may be further evaluated for its abilityto increase or decrease IFN-L polypeptide activity. The measurement ofthe interaction of a test molecule with IFN-L polypeptide may be carriedout in several formats, including cell-based binding assays, membranebinding assays, solution-phase assays, and immunoassays. In general, atest molecule is incubated with an IFN-L polypeptide for a specifiedperiod of time, and IFN-L polypeptide activity is determined by one ormore assays for measuring biological activity.

[0254] The interaction of test molecules with IFN-L polypeptides mayalso be assayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of IFN-L polypeptidescontaining epitope tags as described herein may be used in solution andimmunoassays.

[0255] In the event that IFN-L polypeptides display biological activitythrough an interaction with a binding partner (e.g., a receptor or aligand), a variety of in vitro assays may be used to measure the bindingof an IFN-L polypeptide to the corresponding binding partner (such as aselective binding agent, receptor, or ligand). These assays may be usedto screen test molecules for their ability to increase or decrease therate and/or the extent of binding of an IFN-L polypeptide to its bindingpartner. In one assay, an IFN-L polypeptide is immobilized in the wellsof a microtiter plate. Radiolabeled IFN-L polypeptide binding partner(for example, iodinated IFN-L polypeptide binding partner) and a testmolecule can then be added either one at a time (in either order) orsimultaneously to the wells. After incubation, the wells can be washedand counted for radioactivity, using a scintillation counter, todetermine the extent to which the binding partner bound to the IFN-Lpolypeptide. Typically, a molecule will be tested over a range ofconcentrations, and a series of control wells lacking one or moreelements of the test assays can be used for accuracy in the evaluationof the results. An alternative to this method involves reversing the“positions” of the proteins, i.e., immobilizing IFN-L polypeptidebinding partner to the microtiter plate wells, incubating with the testmolecule and radiolabeled IFN-L polypeptide, and determining the extentof IFN-L polypeptide binding. See, e.g., Current Protocols in MolecularBiology, chap. 18 (Ausubel et al., eds., Green Publishers Inc. and Wileyand Sons 1995).

[0256] As an alternative to radiolabeling, an IFN-L polypeptide or itsbinding partner may be conjugated to biotin, and the presence ofbiotinylated protein can then be detected using streptavidin linked toan enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase(AP), which can be detected colorometrically, or by fluorescent taggingof streptavidin. An antibody directed to an IFN-L polypeptide or to anIFN-L polypeptide binding partner, and which is conjugated to biotin,may also be used for purposes of detection following incubation of thecomplex with enzyme-linked streptavidin linked to AP or HRP.

[0257] A IFN-L polypeptide or an IFN-L polypeptide binding partner canalso be immobilized by attachment to agarose beads, acrylic beads, orother types of such inert solid phase substrates. The substrate-proteincomplex can be placed in a solution containing the complementary proteinand the test compound. After incubation, the beads can be precipitatedby centrifugation, and the amount of binding between an IFN-Lpolypeptide and its binding partner can be assessed using the methodsdescribed herein. Alternatively, the substrate-protein complex can beimmobilized in a column with the test molecule and complementary proteinpassing through the column. The formation of a complex between an IFN-Lpolypeptide and its binding partner can then be assessed using any ofthe techniques described herein (e.g., radiolabelling or antibodybinding).

[0258] Another in vitro assay that is useful for identifying a testmolecule which increases or decreases the formation of a complex betweenan IFN-L polypeptide binding protein and an IFN-L polypeptide bindingpartner is a surface plasmon resonance detector system such as theBIAcore assay system (Pharmacia, Piscataway, N.J.). The BIAcore systemis utilized as specified by the manufacturer. This assay essentiallyinvolves the covalent binding of either IFN-L polypeptide or an IFN-Lpolypeptide binding partner to a dextran-coated sensor chip that islocated in a detector. The test compound and the other complementaryprotein can then be injected, either simultaneously or sequentially,into the chamber containing the sensor chip. The amount of complementaryprotein that binds can be assessed based on the change in molecular massthat is physically associated with the dextran-coated side of the sensorchip, with the change in molecular mass being measured by the detectorsystem.

[0259] In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between an IFN-L polypeptide and an IFN-Lpolypeptide binding partner. In these cases, the assays set forth hereincan be readily modified by adding such additional test compound(s)either simultaneously with, or subsequent to, the first test compound.The remainder of the steps in the assay are as set forth herein.

[0260] In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for an effect on theformation of a complex between an IFN-L polypeptide and IFN-Lpolypeptide binding partner. The assays may be automated to screencompounds generated in phage display, synthetic peptide, and chemicalsynthesis libraries.

[0261] Compounds which increase or decrease the formation of a complexbetween an IFN-L polypeptide and an IFN-L polypeptide binding partnermay also be screened in cell culture using cells and cell linesexpressing either IFN-L polypeptide or IFN-L polypeptide bindingpartner. Cells and cell lines may be obtained from any mammal, butpreferably will be from human or other primate, canine, or rodentsources. The binding of an IFN-L polypeptide to cells expressing IFN-Lpolypeptide binding partner at the surface is evaluated in the presenceor absence of test molecules, and the extent of binding may bedetermined by, for example, flow cytometry using a biotinylated antibodyto an IFN-L polypeptide binding partner. Cell culture assays can be usedadvantageously to further evaluate compounds that score positive inprotein binding assays described herein.

[0262] Cell cultures can also be used to screen the impact of a drugcandidate. For example, drug candidates may decrease or increase theexpression of the IFN-L gene. In certain embodiments, the amount ofIFN-L polypeptide or an IFN-L polypeptide fragment that is produced maybe measured after exposure of the cell culture to the drug candidate. Incertain embodiments, one may detect the actual impact of the drugcandidate on the cell culture. For example, the over-expression of aparticular gene may have a particular impact on the cell culture. Insuch cases, one may test a drug candidate's ability to increase ordecrease the expression of the gene or its ability to prevent or inhibita particular impact on the cell culture. In other examples, theproduction of a particular metabolic product such as a fragment of apolypeptide, may result in, or be associated with, a disease orpathological condition. In such cases, one may test a drug candidate'sability to decrease the production of such a metabolic product in a cellculture.

[0263] Internalizing Proteins

[0264] The tat protein sequence (from HIV) can be used to internalizeproteins into a cell. See, e.g., Falwell et al., 1994, Proc. Natl. Acad.Sci. U.S.A. 91:664-68. For example, an 11 amino acid sequence(Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 18) of the HIV tat protein (termedthe “protein transduction domain,” or TAT PDT) has been described asmediating delivery across the cytoplasmic membrane and the nuclearmembrane of a cell. See Schwarze et al, 1999, Science 285:1569-72; andNagahara et al., 1998, Nat. Med. 4:1449-52. In these procedures,FITC-constructs (FITC-labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO:19), which penetrate tissues following intraperitoneal administration,are prepared, and the binding of such constructs to cells is detected byfluorescence-activated cell sorting (FACS) analysis. Cells treated witha tat-β-gal fusion protein will demonstrate β-gal activity. Followinginjection, expression of such a construct can be detected in a number oftissues, including liver, kidney, lung, heart, and brain tissue. It isbelieved that such constructs undergo some degree of unfolding in orderto enter the cell, and as such, may require a refolding following entryinto the cell.

[0265] It will thus be appreciated that the tat protein sequence may beused to internalize a desired polypeptide into a cell. For example,using the tat protein sequence, an IFN-L antagonist (such as ananti-IFN-L selective binding agent, small molecule, soluble receptor, orantisense oligonucleotide) can be administered intracellularly toinhibit the activity of an IFN-L molecule. As used herein, the term“IFN-L molecule” refers to both IFN-L nucleic acid molecules and IFN-Lpolypeptides as defined herein. Where desired, the IFN-L protein itselfmay also be internally administered to a cell using these procedures.See also, Straus, 1999, Science 285:1466-67.

[0266] Cell Source Identification Using IFN-L Polypeptide

[0267] In accordance with certain embodiments of the invention, it maybe useful to be able to determine the source of a certain cell typeassociated with an IFN-L polypeptide. For example, it may be useful todetermine the origin of a disease or pathological condition as an aid inselecting an appropriate therapy. In certain embodiments, nucleic acidsencoding an IFN-L polypeptide can be used as a probe to identify cellsdescribed herein by screening the nucleic acids of the cells with such aprobe. In other embodiments, one may use anti-IFN-L polypeptideantibodies to test for the presence of IFN-L polypeptide in cells, andthus, determine if such cells are of the types described herein.

[0268] IFN-L Polypeptide Compositions and Administration

[0269] Therapeutic compositions are within the scope of the presentinvention. Such IFN-L polypeptide pharmaceutical compositions maycomprise a therapeutically effective amount of an IFN-L polypeptide oran IFN-L nucleic acid molecule in admixture with a pharmaceutically orphysiologically acceptable formulation agent selected for suitabilitywith the mode of administration. Pharmaceutical compositions maycomprise a therapeutically effective amount of one or more IFN-Lpolypeptide selective binding agents in admixture with apharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration.

[0270] Acceptable formulation materials preferably are nontoxic torecipients at the dosages and concentrations employed.

[0271] The pharmaceutical composition may contain formulation materialsfor modifying, maintaining, or preserving, for example, the pH,osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption, or penetration ofthe composition. Suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine, or lysine), antimicrobials, antioxidants (such as ascorbicacid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates, or other organicacids), bulking agents (such as mannitol or glycine), chelating agents(such as ethylenediamine tetraacetic acid (EDTA)), complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants. See Remington's Pharmaceutical Sciences (18th Ed., A. R.Gennaro, ed., Mack Publishing Company 1990.

[0272] The optimal pharmaceutical composition will be determined by askilled artisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the IFN-L molecule.

[0273] The primary vehicle or carrier in a pharmaceutical compositionmay be either aqueous or non-aqueous in nature. For example, a suitablevehicle or carrier for injection may be water, physiological salinesolution, or artificial cerebrospinal fluid, possibly supplemented withother materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. Other exemplary pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,which may further include sorbitol or a suitable substitute. In oneembodiment of the present invention, IFN-L polypeptide compositions maybe prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington 'sPharnaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the IFN-L polypeptide product may beformulated as a lyophilizate using appropriate excipients such assucrose.

[0274] The IFN-L polypeptide pharmaceutical compositions can be selectedfor parenteral delivery. Alternatively, the compositions may be selectedfor inhalation or for delivery through the digestive tract, such asorally. The preparation of such pharmaceutically acceptable compositionsis within the skill of the art.

[0275] The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

[0276] When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired IFN-L molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which an IFN-L molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads, or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

[0277] In one embodiment, a pharmaceutical composition may be formulatedfor inhalation. For example, IFN-L polypeptide may be formulated as adry powder for inhalation. IFN-L polypeptide or nucleic acid moleculeinhalation solutions may also be formulated with a propellant foraerosol delivery. In yet another embodiment, solutions may be nebulized.Pulmonary administration is further described in PCT Pub. No. WO94/20069, which describes the pulmonary delivery of chemically modifiedproteins.

[0278] It is also contemplated that certain formulations may beadministered orally. In one embodiment of the present invention, IFN-Lpolypeptides that are administered in this fashion can be formulatedwith or without those carriers customarily used in the compounding ofsolid dosage forms such as tablets and capsules. For example, a capsulemay be designed to release the active portion of the formulation at thepoint in the gastrointestinal tract when bioavailability is maximizedand pre-systemic degradation is minimized. Additional agents can beincluded to facilitate absorption of the IFN-L polypeptide. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

[0279] Another pharmaceutical composition may involve an effectivequantity of IFN-L polypeptides in a mixture with non-toxic excipientsthat are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit-dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

[0280] Additional IFN-L polypeptide pharmaceutical compositions will beevident to those skilled in the art, including formulations involvingIFN-L polypeptides in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, e.g. PCT/US93/00829, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions.

[0281] Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 058481), copolymers of L-glutamic acid and ganunaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent No. 133988).Sustained-release compositions may also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European PatentNos. 036676, 088046, and 143949.

[0282] The IFN-L pharmaceutical composition to be used for in vivoadministration typically must be sterile. This may be accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using this method may be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0283] Once the pharmaceutical composition has been formulated, it maybe stored in sterile vials as a solution, suspension, gel, emulsion,solid, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

[0284] In a specific embodiment, the present invention is directed tokits for producing a single-dose administration unit. The kits may eachcontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. Also included within the scopeof this invention are kits containing single and multi-chamberedpre-filled syringes (e.g., liquid syringes and lyosyringes).

[0285] The effective amount of an IFN-L pharmaceutical composition to beemployed therapeutically will depend, for example, upon the therapeuticcontext and objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the IFN-Lmolecule is being used, the route of administration, and the size (bodyweight, body surface, or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 μg/kg up to about 100 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

[0286] The frequency of dosing will depend upon the pharmacokineticparameters of the IFN-L molecule in the formulation being used.Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

[0287] The route of administration of the pharmaceutical composition isin accord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

[0288] Alternatively or additionally, the composition may beadministered locally via implantation of a membrane, sponge, or otherappropriate material onto which the desired molecule has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

[0289] In some cases, it may be desirable to use IFN-L polypeptidepharmaceutical compositions in an ex vivo manner. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to IFN-L polypeptide pharmaceutical compositions after which thecells, tissues, or organs are subsequently implanted back into thepatient.

[0290] In other cases, an IFN-L polypeptide can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete the IFN-Lpolypeptide. Such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. Optionally, the cells may beimmortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the protein product(s) but prevent the destructionof the cells by the patient's immune system or by other detrimentalfactors from the surrounding tissues.

[0291] As discussed herein, it may be desirable to treat isolated cellpopulations (such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like) with one or more IFN-Lpolypeptides. This can be accomplished by exposing the isolated cells tothe polypeptide directly, where it is in a form that is permeable to thecell membrane.

[0292] Additional embodiments of the present invention relate to cellsand methods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally-silent IFN-L gene, or an under-expressedgene, and thereby produce a cell which expresses therapeuticallyefficacious amounts of IFN-L polypeptides.

[0293] Homologous recombination is a technique originally developed fortargeting genes to induce or correct mutations in transcriptionallyactive genes. Kucherlapati, 1989, Prog. in Nucl. Acid Res. & Mol. Biol.36:301. The basic technique was developed as a method for introducingspecific mutations into specific regions of the mammalian genome (Thomaset al., 1986, Cell 44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12;Doetschman et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or tocorrect specific mutations within defective genes (Doetschman et al.,1987, Nature 330:576-78). Exemplary homologous recombination techniquesare described in U.S. Pat. No. 5,272,071; European Patent Nos. 9193051and 505500; PCT/US90/07642, and PCT Pub No. WO 91/09955).

[0294] Through homologous recombination, the DNA sequence to be insertedinto the genome can be directed to a specific region of the gene ofinterest by attaching it to targeting DNA. The targeting DNA is anucleotide sequence that is complementary (homologous) to a region ofthe genomic DNA. Small pieces of targeting DNA that are complementary toa specific region of the genome are put in contact with the parentalstrand during the DNA replication process. It is a general property ofDNA that has been inserted into a cell to hybridize, and therefore,recombine with other pieces of endogenous DNA through shared homologousregions. If this complementary strand is attached to an oligonucleotidethat contains a mutation or a different sequence or an additionalnucleotide, it too is incorporated into the newly synthesized strand asa result of the recombination. As a result of the proofreading function,it is possible for the new sequence of DNA to serve as the template.Thus, the transferred DNA is incorporated into the genome.

[0295] Attached to these pieces of targeting DNA are regions of DNA thatmay interact with or control the expression of an IFN-L polypeptide,e.g., flanling sequences. For example, a promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredIFN-L polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of the desiredIFN-L polypeptide may be achieved not by transfection of DNA thatencodes the IFN-L gene itself, but rather by the use of targeting DNA(containing regions of homology with the endogenous gene of interest)coupled with DNA regulatory segments that provide the endogenous genesequence with recognizable signals for transcription of an IFN-L gene.

[0296] In an exemplary method, the expression of a desired targeted genein a cell (i.e., a desired endogenous cellular gene) is altered viahomologous recombination into the cellular genome at a preselected site,by the introduction of DNA which includes at least a regulatorysequence, an exon, and a splice donor site. These components areintroduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon, and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

[0297] Altered gene expression, as described herein, encompassesactivating (or causing to be expressed) a gene which is normally silent(unexpressed) in the cell as obtained, as well as increasing theexpression of a gene which is not expressed at physiologicallysignificant levels in the cell as obtained. The embodiments furtherencompass changing the pattern of regulation or induction such that itis different from the pattern of regulation or induction that occurs inthe cell as obtained, and reducing (including eliminating) theexpression of a gene which is expressed in the cell as obtained.

[0298] One method by which homologous recombination can be used toincrease, or cause, IFN-L polypeptide production from a cell'sendogenous IFN-L gene involves first using homologous recombination toplace a recombination sequence from a site-specific recombination system(e.g., Cre/loxP, FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol.,5:521-27; Sauer, 1993, Methods Enzymol., 225:890-900) upstream of (i.e.,5′ to) the cell's endogenous genomic IFN-L polypeptide coding region. Aplasmid containing a recombination site homologous to the site that wasplaced just upstream of the genomic IFN-L polypeptide coding region isintroduced into the modified cell line along with the appropriaterecombinase enzyme. This recombinase causes the plasmid to integrate,via the plasmid's recombination site, into the recombination sitelocated just upstream of the genomic IFN-L polypeptide coding region inthe cell line (Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025-29;O'Gorman et al, 1991, Science 251:1351-55). Any flanking sequences knownto increase transcription (e.g., enhancer/promoter, intron,translational enhancer), if properly positioned in this plasmid, wouldintegrate in such a manner as to create a new or modifiedtranscriptional unit resulting in de novo or increased IFN-L polypeptideproduction from the cell's endogenous IFN-L gene.

[0299] A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic IFN-L polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, and translocation) (Sauer,1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993, Methods Enzymol.,225:890-900) that would create a new or modified transcriptional unitresulting in de novo or increased IFN-L polypeptide production from thecell's endogenous IFN-L gene.

[0300] An additional approach for increasing, or causing, the expressionof IFN-L polypeptide from a cell's endogenous IFN-L gene involvesincreasing, or causing, the expression of a gene or genes (e.g.,transcription factors) and/or decreasing the expression of a gene orgenes (e.g., transcriptional repressors) in a manner which results in denovo or increased IFN-L polypeptide production from the cell'sendogenous IFN-L gene. This method includes the introduction of anon-naturally occurring polypeptide (e.g., a polypeptide comprising asite specific DNA binding domain fused to a transcriptional factordomain) into the cell such that de novo or increased IFN-L polypeptideproduction from the cell's endogenous IFN-L gene results.

[0301] The present invention further relates to DNA constructs useful inthe method of altering expression of a target gene. In certainembodiments, the exemplary DNA constructs comprise: (a) one or moretargeting sequences, (b) a regulatory sequence, (c) an exon, and (d) anunpaired splice-donor site. The targeting sequence in the DNA constructdirects the integration of elements (a)-(d) into a target gene in a cellsuch that the elements (b)-(d) are operatively linked to sequences ofthe endogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

[0302] If the sequence of a particular gene is known, such as thenucleic acid sequence of IFN-L polypeptide presented herein, a piece ofDNA that is complementary to a selected region of the gene can besynthesized or otherwise obtained, such as by appropriate restriction ofthe native DNA at specific recognition sites bounding the region ofinterest. This piece serves as a targeting sequence upon insertion intothe cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA,and any additional sequence attached thereto, will act as an Okazakifragment and will be incorporated into the newly synthesized daughterstrand of DNA. The present invention, therefore, includes nucleotidesencoding an IFN-L polypeptide, which nucleotides may be used astargeting sequences.

[0303] IFN-L polypeptide cell therapy, e.g., the implantation of cellsproducing IFN-L polypeptides, is also contemplated. This embodimentinvolves implanting cells capable of synthesizing and secreting abiologically active form of IFN-L polypeptide. Such IFN-Lpolypeptide-producing cells can be cells that are natural producers ofIFN-L polypeptides or may be recombinant cells whose ability to produceIFN-L polypeptides has been augmented by transformation with a geneencoding the desired IFN-L polypeptide or with a gene augmenting theexpression of IFN-L polypeptide. Such a modification may be accomplishedby means of a vector suitable for delivering the gene as well aspromoting its expression and secretion. In order to minimize a potentialimmunological reaction in patients being administered an IFN-Lpolypeptide, as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing IFN-Lpolypeptide be of human origin and produce human IFN-L polypeptide.Likewise, it is preferred that the recombinant cells producing IFN-Lpolypeptide be transformed with an expression vector containing a geneencoding a human IFN-L polypeptide.

[0304] Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of IFN-L polypeptide, but that preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce IFN-L polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

[0305] Techniques for the encapsulation of living cells are known in theart, and the preparation of the encapsulated cells and theirimplantation in patients may be routinely accomplished. For example,Baetge et al. (PCT Pub. No. WO 95/05452 and PCT/US94/09299) describemembrane capsules containing genetically engineered cells for theeffective delivery of biologically active molecules. The capsules arebiocompatible and are easily retrievable. The capsules encapsulate cellstransfected with recombinant DNA molecules comprising DNA sequencescoding for biologically active molecules operatively linked to promotersthat are not subject to down-regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischer et al.). See also, PCT Pub. No. WO 91/10470 (Aebischer etal.); Winn et al., 1991, Exper. Neurol. 113:322-29; Aebischer et al.,1991, Exper. Neurol. 111:269-75; and Tresco et al., 1992, ASAIO38:17-23.

[0306] In vivo and in vitro gene therapy delivery of IFN-L polypeptidesis also envisioned. One example of a gene therapy technique is to usethe IFN-L gene (either genomic DNA, cDNA, and/or synthetic DNA) encodingan IFN-L polypeptide which may be operably linked to a constitutive orinducible promoter to form a “gene therapy DNA construct.” The promotermay be homologous or heterologous to the endogenous IFN-L gene, providedthat it is active in the cell or tissue type into which the constructwill be inserted. Other components of the gene therapy DNA construct mayoptionally include DNA molecules designed for site-specific integration(e.g., endogenous sequences useful for homologous recombination),tissue-specific promoters, enhancers or silencers, DNA molecules capableof providing a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, transcription factorsenhancing expression from-a vector, and factors enabling vectorproduction.

[0307] A gene therapy DNA construct can then be introduced into cells(either ex vivo or in vivo) using viral or non-viral vectors. One meansfor introducing the gene therapy DNA construct is by means of viralvectors as described herein. Certain vectors, such as retroviralvectors, will deliver the DNA construct to the chromosomal DNA of thecells, and the gene can integrate into the chromosomal DNA. Othervectors will function as episomes, and the gene therapy DNA constructwill remain in the cytoplasm.

[0308] In yet other embodiments, regulatory elements can be included forthe controlled expression of the IFN-L gene in the target cell. Suchelements are turned on in response to an appropriate effector. In thisway, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs to dimerize chimeric proteins which contain a smallmolecule-binding domain and a domain capable of initiating a biologicalprocess, such as a DNA-binding protein or transcriptional activationprotein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).The dimerization of the proteins can be used to initiate transcriptionof the transgene.

[0309] An alternative regulation technology uses a method of storingproteins expressed from the gene of interest inside the cell as anaggregate or cluster. The gene of interest is expressed as a fusionprotein that includes a conditional aggregation domain that results inthe retention of the aggregated protein in the endoplasmic reticulum.The stored proteins are stable and inactive inside the cell. Theproteins can be released, however, by administering a drug (e.g., smallmolecule ligand) that removes the conditional aggregation domain andthereby specifically breaks apart the aggregates or clusters so that theproteins may be secreted from the cell. See Aridor et al., 2000, Science287:816-17 and Rivera et al., 2000, Science 287:826-30.

[0310] Other suitable control means or gene switches include, but arenot limited to, the systems described herein. Mifepristone (RU486) isused as a progesterone antagonist. The binding of a modifiedprogesterone receptor ligand-binding domain to the progesteroneantagonist activates transcription by forming a dimer of twotranscription factors that then pass into the nucleus to bind DNA. Theligand-binding domain is modified to eliminate the ability of thereceptor to bind to the natural ligand. The modified steroid hormonereceptor system is further described in U.S. Pat. No. 5,364,791 and PCTPub. Nos. WO 96/40911 and WO 97/10337.

[0311] Yet another control system uses ecdysone (a fruit fly steroidhormone) which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain, DNA-bindingdomain, and ligand-binding domain to initiate transcription. Theecdysone system is further described in U.S. Pat. No. 5,514,578 and PCTPub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.

[0312] Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

[0313] Additional expression control systems and nucleic acid constructsare described in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

[0314] In vivo gene therapy may be accomplished by introducing the geneencoding IFN-L polypeptide into cells via local injection of an IFN-Lnucleic acid molecule or by other appropriate viral or non-viraldelivery vectors. Hefti, 1994, Neurobiology 25:1418-35. For example, anucleic acid molecule encoding an IFN-L polypeptide may be contained inan adeno-associated virus (AAV) vector for delivery to the targetedcells (see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No.PCT/US95/07178). The recombinant AAV genome typically contains AAVinverted terminal repeats flanking a DNA sequence encoding an IFN-Lpolypeptide operably linked to functional promoter and polyadenylationsequences.

[0315] Alternative suitable viral vectors include, but are not limitedto, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. Nos. 5,631,236 (involvingadenoviral vectors), 5,672,510 (involving retroviral vectors), 5,635,399(involving retroviral vectors expressing cytokines).

[0316] Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include induciblepromoters, tissue-specific enhancer-promoters, DNA sequences designedfor site-specific integration, DNA sequences capable of providing aselective advantage over the parent cell, labels to identify transformedcells, negative selection systems and expression control systems (safetymeasures), cell-specific binding agents (for cell targeting),cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as methods of vector manufacture.Such additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. Nos. 4,970,154 (involvingelectroporation techniques), 5,679,559 (describing alipoprotein-containing system for gene delivery), 5,676,954 (involvingliposome carriers), 5,593,875 (describing methods for calcium phosphatetransfection), and 4,945,050 (describing a process wherein biologicallyactive particles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells), and PCT Pub. No. WO 96/40958 (involving nuclearligands).

[0317] It is also contemplated that IFN-L gene therapy or cell therapycan further include the delivery of one or more additionalpolypeptide(s) in the same or a different cell(s). Such cells may beseparately introduced into the patient, or the cells may be contained ina single implantable device, such as the encapsulating membranedescribed above, or the cells may be separately modified by means ofviral vectors.

[0318] A means to increase endogenous IFN-L polypeptide expression in acell via gene therapy is to insert one or more enhancer elements intothe IFN-L polypeptide promoter, where the enhancer elements can serve toincrease transcriptional activity of the IFN-L gene. The enhancerelements used will be selected based on the tissue in which one desiresto activate the gene—enhancer elements known to confer promoteractivation in that tissue will be selected. For example, if a geneencoding an IFN-L polypeptide is to be “turned on” in T-cells, the lckpromoter enhancer element may be used. Here, the functional portion ofthe transcriptional element to be added may be inserted into a fragmentof DNA containing the IFN-L polypeptide promoter (and optionally,inserted into a vector and/or 5′ and/or 3′ flanking sequences) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct,” can then be introduced into the desired cellseither ex vivo or in vivo.

[0319] Gene therapy also can be used to decrease IFN-L polypeptideexpression by modifying the nucleotide sequence of the endogenouspromoter. Such modification is typically accomplished via homologousrecombination methods. For example, a DNA molecule containing all or aportion of the promoter of the IFN-L gene selected for inactivation canbe engineered to remove and/or replace pieces of the promoter thatregulate transcription. For example, the TATA box and/or the bindingsite of a transcriptional activator of the promoter may be deleted usingstandard molecular biology techniques; such deletion can inhibitpromoter activity thereby repressing the transcription of thecorresponding IFN-L gene. The deletion of the TATA box or thetranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofthe IFN-L polypeptide promoter (from the same or a related species asthe IFN-L gene to be regulated) in which one or more of the TATA boxand/or transcriptional activator binding site nucleotides are mutatedvia substitution, deletion and/or insertion of one or more nucleotides.As a result, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. This construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified, may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described herein. Typically, the integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

[0320] Therapeutic Uses

[0321] IFN-L nucleic acid molecules, polypeptides, and agonists andantagonists thereof can be used to treat, diagnose, ameliorate, orprevent a number of diseases, disorders, or conditions, including thoserecited herein.

[0322] IFN-L polypeptide agonists and antagonists include thosemolecules which regulate IFN-L polypeptide activity and either increaseor decrease at least one activity of the mature form of the IFN-Lpolypeptide. Agonists or antagonists may be co-factors, such as aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule, which interact with IFN-L polypeptide and thereby regulate itsactivity. Potential polypeptide agonists or antagonists includeantibodies that react with either soluble or membrane-bound forms ofIFN-L polypeptides that comprise part or all of the extracellulardomains of the said proteins. Molecules that regulate IFN-L polypeptideexpression typically include nucleic acids encoding IFN-L polypeptidethat can act as anti-sense regulators of expression.

[0323] IFN-L polypeptides may play a role in controlling the growth andmaintenance of cancer cells based on the homology of IFN-L polypeptidesto known interferons. Accordingly, IFN-L nucleic acid molecules,polypeptides, and agonists and antagonists thereof may be useful for thediagnosis and/or treatment of cancer. Examples of such cancers include,but are not limited to, chronic myelogenous leukemia, hairy cellleukemia, Kaposi's sarcoma, melanomas, lung cancer, brain cancer, breastcancer, cancers of the hematopoetic system, prostate cancer, ovariancancer, and testicular cancer. Other cancers are encompassed within thescope of the invention

[0324] IFN-L poylpeptides may play a role in the modulation of theimmune system based on the homology of IFN-polypeptides to knowninterferons. Accordingly, IFN-L nucleic acid molecules, polypeptides,and agonists and antagonists thereof may be useful for the diagnosisand/or treatment of dysfunction of the immune system. Examples of suchdiseases include, but are not limited to, multiple sclerosis, rheumatoidarthritis, psioriatic arthritis, inflammatory arthritis, osteoarthritis,inflammatory joint disease, autoimmune disease, lupus, diabetes,inflammatory bowel disease, transplant rejection, and graft vs. hostdisease. Other diseases influenced by the dysfinction of the immunesystem are encompassed within the scope of the invention.

[0325] IFN-L polypeptides may play a role in the control of viral andmicrobial infections based on the homology of IFN-polypeptides to knowninterferons. Accordingly, IFN-L nucleic acid molecules, polypeptides,and agonists and antagonists thereof may be useful for the diagnosisand/or treatment of infections. Examples of such diseases include, butare not limited to, hepatitis, human immunodeficiency virus, humanpapilloma virus, and chronic granulamatous. Other diseases caused byinfections are encompassed within the scope of the invention.

[0326] IFN-L polypeptides may play a role in the control of boneformation and maintenance based on the homology of IFN-polypeptides toknown interferons. Accordingly, IFN-L nucleic acid molecules,polypeptides, and agonists and antagonists may be useful for thediagnosis and/or treatment of bone disorders. Examples of such diseasesinclude, but are not limited to, osteoporosis, osteopetrosis,osteogenesis imperfecta, Paget's disease, periodontal disease, andhypercalcemia. Other bone disorders are encompassed within the scope ofthe invention.

[0327] IFN-L polypeptides may play a role in the inappropriateproliferation of cells based on the homology of IFN-polypeptides toknown interferons. Accordingly, IFN-L nucleic acid molecules,polypeptides, and agonists and antagonists may be useful for thediagnosis and/or treatment of diseases where there is abnormal cellproliferation. Examples of such diseases include, but are not limitedto, arteriosclerosis and vascular restenosis. Other diseases influencedby the inappropriate proliferation of cells are encompassed within thescope of the invention.

[0328] In a specific embodiment, the present invention is directed tothe use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with secreted or soluble humanfas antigen or recombinant versions thereof (PCT Pub. No. WO 96/20206;Mountz et al., 1995, J. Immunol., 155:4829-37; and European Patent No.510691). PCT Pub. No. WO 96/20206 discloses secreted human fas antigen(native and recombinant, including an Ig fusion protein), methods forisolating the genes responsible for coding the soluble recombinant humanfas antigen, methods for cloning the gene in suitable vectors and celltypes, and methods for expressing the gene to produce the inhibitors.European Patent No. 510691 teaches nucleic acids coding for human fasantigen, including soluble fas antigen, vectors expressing for saidnucleic acids, and transformants transfected with the vector. Whenadministered parenterally, doses of a secreted or soluble fas antigenfusion protein each are generally from about 1 μg/kg to about 100 μg/kg.

[0329] Treatment of the diseases and disorders recited herein caninclude the use of first line drugs for control of pain andinflammation; these drugs are classified as non-steroidal,anti-inflammatory drugs (NSAIDs). Secondary treatments includecorticosteroids, slow acting antirheumatic drugs (SAARDs), or diseasemodifying (DM) drugs. Information regarding the following compounds canbe found in The Merck Manual of Diagnosis and Therapy (16th ed. 1992)and in Pharmaprojects (PJB Publications Ltd).

[0330] In a specific embodiment, the present invention is directed tothe use of an IFN-L polypeptide and any of one or more NSAIDs for thetreatment of the diseases and disorders recited herein, including acuteand chronic inflammation such as rheumatic diseases, and graft versushost disease. NSAIDs owe their anti-inflammatory action, at least inpart, to the inhibition of prostaglandin synthesis (Goodman and Gilman,The Pharmacological Basis of Therapeutics (7th ed. 1985)). NSAIDs can becharacterized into at least nine groups: (1) salicylic acid derivatives,(2) propionic acid derivatives, (3) acetic acid derivatives, (4) fenamicacid derivatives, (5) carboxylic acid derivatives, (6) butyric acidderivatives, (7) oxicams, (8) pyrazoles, and (9) pyrazolones.

[0331] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moresalicylic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. Such salicylic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof comprise:acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate, magnesium salicylate,choline salicylate, diflusinal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide O-acetic acid, salsalate, sodium salicylate andsulfasalazine. Structurally related salicylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0332] In an additional specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more propionic acid derivatives; prodrug esters, or pharmaceuticallyacceptable salts thereof. The propionic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof comprise:alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen,fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen,ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen,ketoprofen, loxoprofen, miroprofen, naproxen, naproxen sodium,oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinicacid, pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen.Structurally related propionic acid derivatives having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group.

[0333] In yet another specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more acetic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. The acetic acid derivatives, prodrug esters,and pharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac potassium, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium,zidometacin and zomepirac. Structurally related acetic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

[0334] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more fenamicacid derivatives, prodrug esters, or pharmaceutically acceptable saltsthereof. The fenamic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: enfenamic acid,etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamatesodium, medofenamic acid, mefenamic acid, niflumic acid, talniflumate,terofenamate, tolfenamic acid and ufenamate. Structurally relatedfenamic acid derivatives having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

[0335] In an additional specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more carboxylic acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof. The carboxylic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof which can be usedcomprise: clidanac, diflunisal, flufenisal, inoridine, ketorolac andtinoridine. Structurally related carboxylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0336] In yet another specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more butyric acid derivatives, prodrug esters, or pharmaceuticallyacceptable salts thereof The butyric acid derivatives, prodrug esters,and pharmaceutically acceptable salts thereof comprise:

[0337] bumadizon, butibufen, fenbufen and xenbucin. Structurally relatedbutyric acid derivatives having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

[0338] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreoxicams, prodrug esters, or pharmaceutically acceptable salts thereofThe oxicams, prodrug esters, and pharmaceutically acceptable saltsthereof comprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam,tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide4-(N-phenyl)-carboxamide. Structurally related oxicams having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

[0339] In still another specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more pyrazoles, prodrug esters, or pharmaceutically acceptable saltsthereof. The pyrazoles, prodrug esters, and pharmaceutically acceptablesalts thereof which may be used comprise: difenamizole and epirizole.Structurally related pyrazoles having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

[0340] In an additional specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment or, concurrent treatment) with any of oneor more pyrazolones, prodrug esters, or pharmaceutically acceptablesalts thereof. The pyrazolones, prodrug esters, and pharmaceuticallyacceptable salts thereof which may be used comprise: apazone,azapropazone, benzpiperylon, feprazone, mofebutazone, morazone,oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone,ramifenazone, suxibuzone and thiazolinobutazone. Structurally relatedpyrazalones having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

[0341] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more of thefollowing: NSAIDs: ε-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine,bendazac, bendazac lysinate, benzydamine, beprozin, broperamole,bucolome, bufezolac, ciproquazone, cloximate, dazidamine, deboxamet,detomidine, difenpiramide, difenpyramide, difisalamine, ditazol,emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole,flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene,isonixim, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysinclonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein,orpanoxin, oxaceprol, oxapadol, paranyline, perisoxal, perisoxalcitrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone,proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol,tryptamid and those designated by company code number such as 480156S,AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C,CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182,KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102,PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281,SY6001, TA60, TAI-901 (4-benzoyl-1-indancarboxylic acid), TVX2706,U60257, UR2301 and WY41770. Structurally related NSAIDs having similaranalgesic and anti-inflammatory properties to the NSAIDs are alsointended to be encompassed by this group.

[0342] In still another specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment or concurrent treatment) with any of oneor more corticosteroids, prodrug esters, or pharmaceutically acceptablesalts thereof for the treatment of the diseases and disorders recitedherein, including acute and chronic inflammation such as rheumaticdiseases, graft versus host disease, and multiple sclerosis.Corticosteroids, prodrug esters, and pharmaceutically acceptable saltsthereof include hydrocortisone and compounds which are derived fromhydrocortisone, such as 21-acetoxypregnenolone, alclomerasone,algestone, amcinonide, beclomethasone, betamethasone, betamethasonevalerate, budesonide, chloroprednisone, clobetasol, clobetasolpropionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol,corticosterone, cortisone, cortivazol, deflazacon, desonide,desoximerasone, dexamethasone, diflorasone, diflucortolone,difluprednate, enoxolone, fluazacort, flucloronide, flumethasone,flumethasone pivalate, flucinolone acetonide, flunisolide, fluocinonide,fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluocortolonehexanoate, diflucortolone valerate, fluorometholone, fluperoloneacetate, fluprednidene acetate, fluprednisolone, flurandenolide,formocortal, halcinonide, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisonebutyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0343] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreslow-acting antirheumatic drugs (SAARDs) or disease modifyingantirheumatic drugs (DMARDS), prodrug esters, or pharmaceuticallyacceptable salts thereof for the treatment of the diseases and disordersrecited herein, including acute and chronic inflammation such asrheumatic diseases, graft versus host disease, and multiple sclerosis.SAARDs or DMARDS, prodrug esters, and pharmaceutically acceptable saltsthereof comprise: allocupreide sodium, auranofin, aurothioglucose,aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine,clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone,15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinegold salt, gold sodium thiomalate, gold sodium thiosulfate),hydroxychloroquine, hydroxychloroquine sulfate, hydroxyurea, kebuzone,levamisole, lobenzarit, melittin, 6-mercaptopurine, methotrexate,mizoribine, mycophenolate mofetil, myoral, nitrogen mustard,D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580,rapamycin, thiols, thymopoietin and vincristine. Structurally relatedSAARDs or DMARDs having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

[0344] In another specific embodiment, the present invention is directedto the use of an IFN-L polypeptide in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more COX2inhibitors, prodrug esters, or pharmaceutically acceptable salts thereoffor the treatment of the diseases and disorders recited herein,including acute and chronic inflammation. Examples of COX2 inhibitors,prodrug esters, or pharmaceutically acceptable salts thereof include,for example, celecoxib. Structurally related COX2 inhibitors havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0345] In still another specific embodiment, the present invention isdirected to the use of an IFN-L polypeptide in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more antimicrobials, prodrug esters, or pharmaceutically acceptablesalts thereof for the treatment of the diseases and disorders recitedherein, including acute and chronic inflammation. Antimicrobialsinclude, for example, the broad classes of penicillins, cephalosporinsand other beta-lactams, aminoglycosides, azoles, quinolones, macrolides,rifamycins, tetracyclines, sulfonamides, lincosamides and polymyxins.The penicillins include, but are not limited to, penicillin G,penicillin V, methicillin, nafcillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, ampicillin, ampicillin/sulbactam,amoxicillin, amoxicillin/clavulanate, hetacillin, cyclacillin,bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin,ticarcillin/clavulanate, azlocillin, mezlocillin, peperacillin, andmecillinam. The cephalosporins and other beta-lactams include, but arenot limited to, cephalothin, cephapirin, cephalexin, cephradine,cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefoxitin,ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime, moxalactam,ceftizoxime, cetriaxone, cephoperazone, ceftazidime, imipenem andaztreonam. The aminoglycosides include, but are not limited to,streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycinand neomycin. The azoles include, but are not limited to, fluconazole.The quinolones include, but are not limited to, nalidixic acid,norfloxacin, enoxacin, ciprofloxacin, ofioxacin, sparfloxacin andtemafloxacin. The macrolides include, but are not limited to,erythomycin, spiramycin and azithromycin. The rifamycins include, butare not limited to, rifampin. The tetracyclines include, but are notlimited to, spicycline, chlortetracycline, clomocycline, demeclocycline,deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,minocycline, oxytetracycline, penimepicycline, pipacycline,rolitetracycline, sancycline, senociclin and tetracycline. Thesulfonamides include, but are not limited to, sulfanilamide,sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole andco-trimoxazole (trimethoprim/sulfamethoxazole). The lincosamidesinclude, but are not limited to, clindamycin and lincomycin. Thepolymyxins (polypeptides) include, but are not limited to, polymyxin Band colistin.

[0346] Agonists or antagonists of IFN-L polypeptide function may be used(simultaneously or sequentially) in combination with one or morecytokines, growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the condition beingtreated.

[0347] Other diseases caused by or mediated by undesirable levels ofIFN-L polypeptides are encompassed within the scope of the invention.Undesirable levels include excessive levels of IFN-L polypeptides andsub-normal levels of IFN-L polypeptides.

[0348] Uses of IFN-L Nucleic Acids and Polypeptides

[0349] Nucleic acid molecules of the invention (including those that donot themselves encode biologically active polypeptides) may be used tomap the locations of the IFN-L gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

[0350] IFN-L nucleic acid molecules (including those that do notthemselves encode biologically active polypeptides), may be useful ashybridization probes in diagnostic assays to test, either qualitativelyor quantitatively, for the presence of an IFN-L nucleic acid molecule inmammalian tissue or bodily fluid samples.

[0351] Other methods may also be employed where it is desirable toinhibit the activity of one or more IFN-L polypeptides. Such inhibitionmay be effected by nucleic acid molecules that are complementary to andhybridize to expression control sequences (triple helix formation) or toIFN-L mRNA. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of an IFN-L genecan be introduced into the cell. Anti-sense probes may be designed byavailable techniques using the sequence of the IFN-L gene disclosedherein. Typically, each such antisense molecule will be complementary tothe start site (5′ end) of each selected IFN-L gene. When the antisensemolecule then hybridizes to the corresponding IFN-L mRNA, translation ofthis mRNA is prevented or reduced. Anti-sense inhibitors provideinformation relating to the decrease or absence of an IFN-L polypeptidein a cell or organism.

[0352] Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more IFN-L polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected IFN-Lpolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

[0353] In addition, an IFN-L polypeptide, whether biologically active ornot, may be used as an immunogen, that is, the polypeptide contains atleast one epitope to which antibodies may be raised. Selective bindingagents that bind to an IFN-L polypeptide (as described herein) may beused for in vivo and in vitro diagnostic purposes, including, but notlimited to, use in labeled form to detect the presence of IFN-Lpolypeptide in a body fluid or cell sample. The antibodies may also beused to prevent, treat, or diagnose a number of diseases and disorders,including those recited herein. The antibodies may bind to an IFN-Lpolypeptide so as to diminish or block at least one activitycharacteristic of an IFN-L polypeptide, or may bind to a polypeptide toincrease at least one activity characteristic of an IFN-L polypeptide(including by increasing the pharmacokinetics of the IFN-L polypeptide).

[0354] The IFN-L polypeptides of the present invention can be used toclone IFN-L polypeptide receptors, using an expression cloning strategy.Radiolabeled (¹²⁵Iodine) IFN-L polypeptide or affinity/activity-taggedIFN-L polypeptide (such as an Fc fusion or an alkaline phosphatasefusion) can be used in binding assays to identify a cell type or cellline or tissue that expresses IFN-L polypeptide receptors. RNA isolatedfrom such cells or tissues can be converted to cDNA, cloned into amammalian expression vector, and transfected into mammalian cells (suchas COS or 293 cells) to create an expression library. A radiolabeled ortagged IFN-L polypeptide can then be used as an affinity ligand toidentify and isolate from this library the subset of cells that expressthe IFN-L polypeptide receptors on their surface. DNA can then beisolated from these cells and transfected into mammalian cells to createa secondary expression library in which the fraction of cells expressingIFN-L polypeptide receptors is many-fold higher than in the originallibrary. This enrichment process can be repeated iteratively until asingle recombinant clone containing an IFN-L polypeptide receptor isisolated. Isolation of the IFN-L polypeptide receptors is useful foridentifying or developing novel agonists and antagonists of the IFN-Lpolypeptide signaling pathway. Such agonists and antagonists includesoluble IFN-L polypeptide receptors, anti-IFN-L polypeptide receptorantibodies, small molecules, or antisense oligonucleotides, and they maybe used for treating, preventing, or diagnosing one or more of thediseases or disorders described herein.

[0355] A deposit of cDNA encoding human IFN-L polypeptide, subclonedinto pSPORT1 (Gibco BRL) and transfected into E. coli strain DH10B,having Accession No. PTA-976, were made with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 on Nov.23, 1999.

[0356] The following examples are intended for illustration purposesonly, and should not be construed as limiting the scope of the inventionin any way.

EXAMPLE 1 Cloning of the Rat IFN-L Polypeptide Gene

[0357] Generally, materials and methods as described in Sambrook et al.supra were used to clone and analyze the gene encoding rat IFN-Lpolypeptide.

[0358] Sequences encoding the rat IFN-L polypeptide were isolated from arat placenta cDNA library by large scale random cDNA sequencing incombination with computer-assisted analysis. To construct the ratplacenta cDNA library, rat embryo day 17 [E17] placenta mRNA wasprepared by standard methods (Chomczynski and Sacchi, 1987, Anal.Biochem. 162:156). Following synthesis using the Superscript PlasmidcDNA kit (Gibco BRL), rat cDNA was subcloned into the Sal I and Not Isites of the pSPORT1 vector (Gibco BRL).

[0359] Sequence analysis of the full-length cDNA for rat IFN-Lpolypeptide indicated that the gene comprises a 573 bp open readingframe encoding a protein of 191 amino acids (FIG. 1A-1B). The rat IFN-Lpolypeptide sequence is predicted to contain a signal peptide (FIG. 1A,predicted signal peptide indicated by underline). The rat IFN-Lpolypeptide sequence was identified as being a novel member of theinterferon family of proteins following comparisons of the rat IFN-Lpolypeptide sequence with protein sequences in the GenBank database.

EXAMPLE 2 Cloning of the Human IFN-L Polypeptide Gene

[0360] Generally, materials and methods as described in Sambrook et al.supra were used to clone and analyze the gene encoding human IFN-Lpolypeptide.

[0361] An examination of the genomic structure of known members of theInterferon gene family revealed that members of this family share aunique intronless structure. Sequences encoding the human IFN-Lpolypeptide were, therefore, isolated by screening a human genomic DNAlibrary with a probe derived from the rat IFN-L polypeptide gene.

[0362] A radioactive rat IFN-L probe was generated by polymerase chainreaction (PCR) amplification of rat IFN-L polypeptide cDNA. Polymerasechain reactions (PCR) were performed using a Perkin-Elmer 9600thermocycler (PE Biosystems, Foster City, Calif.) and the followingreaction conditions: 20 ng of rat IFN-L polypeptide cDNA, 20 pmol eachof primers 1795-01 (5′-A-T-G-A-C-A-C-T-G-A-A-G-T-A-T-T-T-A-T-G-G-3′; SEQID NO: 20) and 1795-02 (5′-A-T-T-C-A-T-G-T-T-G-A-G-T-A-G-T-T-T-G-T-A-3′;SEQ ID NO: 21), 1 mmol each of dATP, dTTP, dGTP, 0.01 mmol dCTP, 100 μCi³²P-dCTP, 4 mM MgCl₂, 1X PCR buffer, and 5U Taq polymerase (PEBiosystems). A “cold” PCR reaction (i.e., one not performed in thepresence of radioactively labeled dCTP, and utilizing a balanced dNTPmix) was prepared simultaneously with the labeled reaction.Amplification reactions were carried out at 94° C. for 30 seconds, 60°C. for 30 seconds, and 72° C. for 1 minute for 45 cycles. Pooled labeledand unlabeled probe was purified using a Quick Spin G-50 column(Qiagen), boiled at 100° C. for 10 minutes, and chilled on ice for 20minutes prior to addition to the hybridization solution. Probes with aspecific activity of at least 5×10⁵ cpm/μL were generated using thismethod.

[0363] Sequences encoding the human IFN-L polypeptide were isolated byscreening a human lambda genomic DNA library (Stratagene, Cat. No.946206). For the primary screen, 1×10⁶ clones were plated at a densityof 50,000 colonies/plate and transferred to nitrocellulose filters usingstandard techniques. Positive clones were re-screened prior to analysis.

[0364] The rat IFN-L probe was hybridized to the filters overnight at42° C. in 30% formamide, 5X SSC, 2X Denhart's, 10 μg/mL salmon spermDNA, 0.2% SDS, 2 mM EDTA, and 0.1% pyrophosphate. Followinghybridization, filters were washed for 30-60 minutes at room temperaturein 1X SSC and 0.1% SDS and then for 15 minutes at 55° C. in 0.2X SSC and0.1% SDS.

[0365] Three positive clones were recovered following primary andsecondary screening, and lambda phage DNA was prepared by a solid plateculture method. The Not I insert was excised from the clones and ligatedinto pSPORT1 (Gibco BRL), and these ligations were subsequently used totransform E. coli strain DH10. Following transformation, plasmids wererecovered using a Spin Column plasmid prep kit (Qiagen).

[0366] Plasmids derived from the three positive genomic DNA clones wereanalyzed by Southern blot analysis using the rat IFN-L probe utilized inthe genomic DNA library screening. After digesting the recovered plasmidDNA with Hind III, the digested fragments were resolved on an agarosegel, and then transferred to a nylon membrane. Hybridization conditionswere identical to those utilized in the genomic DNA library screen.Southern blot analysis indicated that the three positive genomic cloneswere likely to contain identical genomic inserts. The fragmentshybridizing with the rat IFN-L probe were subsequently subcloned intopSPORT1 for sequencing analysis. This analysis confirmed that the threepositive genomic DNA clones contained identical genomic inserts.

[0367] Sequence analysis of the three genomic clones containingsequences encoding human IFN-L polypeptide indicated that the genecomprises a 621 bp open reading frame encoding a protein of 207 aminoacids (FIGS. 2A-2B). The human IFN-L polypeptide sequence is predictedto contain a signal peptide (FIG. 2A, predicted signal peptide indicatedby underline). Sequence analysis of IFN-L polypeptide strongly suggeststhat the protein is a secreted cytokine molecule.

[0368] A similarity of 64% was observed between the open reading frameof the human IFN-L gene and that of the rat IFN-L cDNA. FIG. 3illustrates the amino acid sequence alignment of human IFN-L polypeptide(SEQ ID NO: 2), human IFN-p (SEQ ID NO: 7), and rat IFN-L polypeptide(SEQ ID NO: 4). Human IFN-L polypeptide is 30% identical to human IFN-p.Human IFN-L polypeptide is 40.5% identical to and 50% similar to ratIFN-L polypeptide. All five predicted cysteine residues in human IFN-Lpolypeptide are perfectly aligned with those in rat IFN-L polypeptide.

EXAMPLE 3 IFN-L mRNA Expression

[0369] Developmental expression patterns of IFN-L mRNA were determinedby Northern blot analysis using a ³²P-labeled full-length rat cDNA probeto detect the presence of the IFN-L polypeptide transcript in severaldifferent stages of mouse and rat embryos. RNA was isolated from the ratand mouse embryos using the same techniques employed for theconstruction of the rat placenta cDNA library. Northern blots wereprehybridized in 40% formamide, 5X SSC, 1 mM EDTA, and 0.1% for 4 hoursat 42° C. The blots were hybridized overnight at 42° C. in the samesolution, except for the addition of the rat IFN-L probe. Followinghybridization, blots were washed for 30 minutes at 60° C. in 1X SSC and0.1% SDS.

[0370] Expression of IFN-L mRNA was examined in various human tissues byRT-PCR using standard techniques. Human IFN-L mRNA was detected inpancreas, small intestine, prostrate, uterus, thyroid, and placenta. Theexpression of IFN-L mRNA is localized by in situ hybridization. A panelof normal embryonic and adult mouse tissues is fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Sectionedtissues are permeabilized in 0.2 M HCl, digested with Proteinase K, andacetylated with triethanolamine and acetic anhydride. Sections areprehybridized for 1 hour at 60° C. in hybridization solution (300 mMNaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1X Denhardt's solution, 0.2%SDS, 10 mM DTT, 0.25 mg/ml tRNA, 25 μg/ml polyA, 25 μg/ml polyc and 50%formamide) and then hybridized overnight at 60° C. in the same solutioncontaining 10% dextran and 2×10 ⁴cpm/μl of a ³³P-labeled antisenseriboprobe complementary to the human IFN-L gene. The riboprobe isobtained by in vitro transcription of a clone containing human IFN-LcDNA sequences using standard techniques.

[0371] Following hybridization, sections are rinsed in hybridizationsolution, treated with RNaseA to digest unhybridized probe, and thenwashed in 0.1X SSC at 55° C. for 30 minutes. Sections are then immersedin NTB-2 emulsion (Kodak, Rochester, N.Y.), exposed for 3 weeks at 4°C., developed, and counterstained with hematoxylin and eosin. Tissuemorphology and hybridization signal are simultaneously analyzed bydarkfield and standard illumination for brain (one sagittal and twocoronal sections), gastrointestinal tract (esophagus, stomach, duodenum,jejunum, ileum, proximal colon, and distal colon), pituitary, liver,lung, heart, spleen, thymus, lymph nodes, kidney, adrenal, bladder,pancreas, salivary gland, male and female reproductive organs (ovary,oviduct, and uterus in the female; and testis, epididymus, prostate,seminal vesicle, and vas deferens in the male), BAT and WAT(subcutaneous, peri-renal), bone (femur), skin, breast, and skeletalmuscle.

EXAMPLE 4 Production of IFN-L Polypeptides

[0372] A. Expression of IFN-L Polypeptides in Bacteria

[0373] PCR was used to amplify template DNA sequences encoding eitherhuman or rat IFN-L polypeptide using primers that corresponded to the 5′and 3′ ends of the sequence (Table I) and which incorporated restrictionenzyme sites to permit insertion of the amplified product into anexpression vector. Following amplification, PCR products were gelpurified, digested with the appropriate restriction enzymes, and ligatedinto the expression vector pAMG21 (ATCC No. 98113) using standardrecombinant DNA techniques. After the ligation of PCR insert and vectorsequences, the ligation reaction mixtures were used to transform an E.coli host strain (e.g., Amgen strain #2596) by electroporation andtransformants were selected for kanamycin drug resistance. Plasmid DNAfrom selected colonies was isolated and subjected to DNA sequencing toconfirm the presence of an appropriate insert.

[0374] To construct a rat IFN-L polypeptide bacterial expression vector,IFN-L nucleic acid sequences were amplified from a cDNA template usingthe primers 1825-22 and 1825-21. The PCR product that was obtainedfollowing amplification with these primers was inserted into the Nde Iand Bam HI sites of pAMG21, and the ligation reaction was then used inbacterial transformation.

[0375] The resulting bacterial clone was designated Amgen strain #3729.FIG. 4 illustrates the nucleotide sequence of the pAMG21 insert of Amgenstrain #3729 and the predicted amino acid sequence encoded by thisinsert.

[0376] A rat IFN-L polypeptide bacterial expression vector, in which thecysteine at position 180 was substituted with a serine residue, wasconstructed using the primers 1825-22 and 1909-56. The PCR product thatwas obtained following amplification with these primers was insertedinto the Nde I and Bam HI sites of pAMG21, and the ligation reaction wasthen used in bacterial transformation. TABLE I SEQ ID Oligonucleotide IDSequence 22 1825-225′-GAATAACATATGTGTGTATATCTCGATCATACTATCTTGGAGAATATG-3′ 23 1825-215′-CCGCGGATCCATTAATTCATGTTCAGCAGTTTGTAAAAAATACTGAAACAACGACGAATTTCC-3′ 241909-565′-CCGCGGATCCATTAATTCATGTTCAGCAGTTTGTAAAAAATACTGAAAGAACGACGAATTTCC-3′ 251967-325′-TTGATCTAGAAAGGAGGAATAACATATGTGTAACCTGCTGAACGTTCACCTGCGTCGTGTTACCTGG-3′26 1982-145′-CCGCGGATCCATTATTTACGACGGAACAGAGCGGTAAATTTGTAAAAGTAGTACAGGCAACGACGATTTCC-3′27 1967-335′-CCGCGGATCCATTATTTACGACGGAACAGAGCGGTAAATTTGTAAAAGTAGTACAGAGAACGACGGATTTCC-3′28 2103-87 5′-AAGGAGCATATGCTGGACTGTAACCTGCTGAACGTTCAC-3′ 29 1200-545′-GTTATTGCTCAGCGGTGGCA-3′

[0377] The resulting bacterial clone was designated Amgen strain #3858.FIG. 5 illustrates the nucleotide sequence of the pAMG21 insert of Amgenstrain #3858 and the predicted amino acid sequence encoded by thisinsert.

[0378] To construct a human IFN-L polypeptide bacterial expressionvector, IFN-L nucleic acid sequences were amplified from a cDNA templateusing the primers 1967-32 and 1982-14. The PCR product that was obtainedfollowing amplification with these primers was inserted into the Xba Iand Bam HI sites of pAMG21, and the ligation reaction was then used inbacterial transformation. The resulting bacterial clone was designatedAmgen strain #4047. FIG. 6 illustrates the nucleotide sequence of thepAMG21 insert of Amgen strain #4047 and the predicted amino acidsequence encoded by this insert.

[0379] A human IFN-L polypeptide bacterial expression vector, in whichthe cysteine at position 193 was substituted with a serine residue, wasconstructed using the primers 1967-32 and 1967-33. The PCR product thatwas obtained following amplification with these primers was insertedinto the Xba I and Bam HI sites of pAMG21, and the ligation reaction wasthen used in bacterial transformation. The resulting bacterial clone wasdesignated Amgen strain #3969. FIG. 7 illustrates the nucleotidesequence of the pAMG21 insert of Amgen strain #3969 and the predictedamino acid sequence encoded by this insert.

[0380] A human IFN-L polypeptide bacterial expression vector, expressingan N-terminal variant of human IFN-L polypeptide, was constructed byamplifying plasmid from strain #4047 with the primers 1967-32 and1967-33. The PCR product that was obtained following amplification withthese primers was inserted into the Nde I and Bam HI sites of pAMG21,and the ligation reaction was then used in bacterial transformation. Theresulting bacterial clone was designated Amgen strain #4182. FIG. 8illustrates the nucleotide sequence of the pAMG21 insert of Amgen strain#4182 and the predicted amino acid sequence encoded by this insert.

[0381] To generate IFN-L polypeptides, transformed host cells were firstincubated in Terrific Broth medium containing 50 μg/mL kanamycin at 30°C. prior to induction of IFN-L polypeptide. Expression of IFN-Lpolypeptide was induced by the addition of 30 ng/mLN-(3-oxohexanoyl)-dl-homoserine lactone followed by a six hourincubation at either 30° C. or 37° C. Expression of IFN-L polypeptidewas evaluated by centrifugation of the culture, resuspension and lysisof the bacterial pellets, and analysis of host cell proteins bySDS-polyacrylamide gel electrophoresis.

[0382] A single band on an SDS polyacrylamide gel corresponding to E.coli produced IFN-L polypeptide was excised from the gel and N-terminalamino acid sequence was determined essentially as described byMatsudaira et al., 1987, J. Biol. Chem. 262:10-35).

[0383] IFN-L polypeptides were purified as follows. Cells were firstlysed in water by high pressure homogenization and inclusion bodies wereharvested by centrifugation. Solubilized inclusion bodies were thensubjected to a variety of refold conditions.

[0384] B. Construction of IFN-L Polypeptide Mammalian Expression Vectors

[0385] Native protein and native protein-Fc fusion versions of bothhuman and rat IFN-L polypeptides were produced in either a CHO or 293mammalian expression system. Template DNA sequences encoding IFN-Lpolypeptide were amplified by PCR using primers corresponding to the 5′and 3′ ends (Table II).

[0386] To construct IFN-L polypeptide expression vectors, IFN-L nucleicacid sequences were amplified as described below. Rat IFN-L nucleic acidsequences were obtained using one of three primer pairs (the forwardprimer 1847-77 and either 1847-88, 1896-56, or 1896-57). A rat IFN-Lpolypeptide-Fc fusion construct was generated by cloning PCR productsprepared with the first set of primers, which incorporated Hind III andNot I cloning sites and no stop codon. Rat IFN-L soluble polypeptideswere generated by cloning PCR products prepared with the second set ofprimers, which incorporated Hind III and Sal I cloning sites and twostop codons, into pDSRα, or the third set of primers, which incorporatedHind III and Not I cloning sites and two stop codons, into pCEP4. TABLEII SEQ ID Oligonucleotide ID Sequence 30 1847-775′-CCCAAGCTTACCATGACACTGAAGTATTTATG-3′ 31 1847-785′-AAGGAAAAAAGCGGCCGCATTCATGTTGAGTAG-3′ 32 1896-565′-ACGCGTCGACTCATCAATTCATGTTGAGTAGTTTG-3′ 33 1896-575′-AAGGAAAAAAGCGGCCGCTCATCAATTCATGTTGAGTAG-3′ 34 1954-455′-ACGCGTCGACTTATTATTTCCTCCTGAATAG-3′ 35 1954-465′-AAGGAAAAAAGCGGCCGCTTATTATTTCCTCCTGAATAGAGC-3′ 36 1955-445′-CCCAAGCTTACCATGAGCACCAAACCTGATATG-3′ 37 1954-475′-CCCAAGCTTACCATGATTCAAAAGTGTTTGTGGC-3′ 38 1954-485′-AAGGAAAAAAGCGGCCGCGCGGCCCTCGATTTTCCTCCTGAATAGAGCTGTAA-3′ 39 1954-495′-AAGGAAAAAAGCGGCCGCTTTCCTCCTGAATAGAGCTGTAA-3′

[0387] Human IFN-L nucleic acid sequences were obtained using one ofthree primer pairs (the forward primer 1954-48 and 1954-49 and theforward primer 1955-44 and either 1854-45 or 1854-46). A human IFN-Lpolypeptide-Fc fusion construct was generated by cloning PCR productsprepared with the first set of primers, which incorporated Not I cloningsites, no stop codon, and a Factor Xa cleavage site. Human IFN-L solublepolypeptides were generated by cloning PCR products prepared with thesecond set of primers, which incorporated Hind III and Sal I cloningsites and two stop codons, into pDSRα, or the third set of primers,which incorporated Hind III and Not I cloning sites and two stop codons,into pCEP4. A second forward primer (1954-47) was also utilized in placeof 1955-44 to generate constructs possessing two initiation codons.

[0388] PCR amplifications were performed using a Perkin-Elmer 9600thermocycler and the following reaction conditions: 20 ng of rat orhuman IFN-L polypeptide cDNA, 20 pmol each of the appropriate primers, 1mmol of dNTPs, 4 mM MgCl₂, 1X PCR buffer, and 5U Taq polymerase (PEBiosystems). Amplification reactions were carried out at 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 1 minute for 4 cyclesfollowed by 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for1 minute for 26 cycles.

[0389] PCR products were purified using Qiagen PCR purification spincolumns and then subjected to digestion with the appropriate restrictionendonucleases. Following digestion, fragments were separated on agarosegels, purified using Qiagen gel purification spin columns, and ligatedinto the appropriate vectors. Ligations were transformed into the E.coli strain DH10. Following sequence analysis of selected transformants,large-scale plasmid stocks were prepared for tissue culturetransfection.

[0390] C. Expression and Purification of IFN-L Polypeptide in MammalianCells

[0391] IFN-L polypeptide expression constructs were introduced into 293EBNA or CHO cells using either a lipofection or calcium phosphateprotocol.

[0392] To conduct functional studies on the IFN-L polypeptides that wereproduced, large quantities of conditioned media were generated from apool of hygromycin selected 293 EBNA clones. The cells were cultured in500 cm Nunc Triple Flasks to 80% confluence before switching to serumfree media a week prior to harvesting the media. Conditioned media washarvested and frozen at −20° C. until purification.

[0393] Conditioned media was purified by affinity chromatography asdescribed below. The media was thawed and then passed through a 0.2 μmfilter. A Protein G column was equilibrated with PBS at pH 7.0, and thenloaded with the filtered media. The column was washed with PBS until theabsorbance at A₂₈₀ reached a baseline. IFN-L polypeptide was eluted fromthe column with 0.1 M Glycine-HCl at pH 2.7 and immediately neutralizedwith 1 M Tris-HCl at pH 8.5. Fractions containing IFN-L polypeptide werepooled, dialyzed in PBS, and stored at −70° C.

[0394] For Factor Xa cleavage of the human IFN-L polypeptide-Fc fusionpolypeptide, affinity chromatography-purified protein was dialyzed in 50mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂ at pH 8.0. The restriction proteaseFactor Xa was added to the dialyzed protein at 1/100 (w/w) and thesample digested overnight at room temperature.

EXAMPLE 5 Biological Activity of IFN-L Polypeptides

[0395] The phosphorylation of IFN-L polypeptide was assayed as follows.Cell lines were exposed to 1 μg/mL of the rat IFN-L Fc fusionpolypeptide generated in Example 4C or to a control solution at 37° C.for 15 minutes. Following IFN-L polypeptide exposure, the cells werelysed and cellular proteins were recovered and separated by SDS-PAGE.The separated proteins were then analyzed by Western blot using ananti-pTyr antibody. Several cell lines showed an increase in cellularprotein phosphorylation following exposure to IFN-L Fc fusionpolypeptide.

EXAMPLE 6 Production of Anti-IFN-L Polypeptide Antibodies

[0396] Antibodies to IFN-L polypeptides may be obtained by immunizationwith purified protein or with IFN-L peptides produced by biological orchemical synthesis. Suitable procedures for generating antibodiesinclude those described in Hudson and Bay, Practical Immunology (2nded., Blackwell Scientific Publications).

[0397] In one procedure for the production of antibodies, animals(typically mice or rabbits) are injected with an IFN-L antigen (such asan IFN-L polypeptide), and those with sufficient serum titer levels asdetermined by ELISA are selected for hybridoma production. Spleens ofimmunized animals are collected and prepared as single cell suspensionsfrom which splenocytes are recovered. The splenocytes are fused to mousemyeloma cells (such as Sp2/0-Agl4 cells), are first incubated in DMEMwith 200 U/mL penicillin, 200 μg/mL streptomycin sulfate, and 4 mMglutamine, and are then incubated in HAT selection medium (hypoxanthine,aminopterin, and thymidine). After selection, the tissue culturesupernatants are taken from each fusion well and tested for anti-IFN-Lantibody production by ELISA.

[0398] Alternative procedures for obtaining anti-IFN-L antibodies mayalso be employed, such as the immunization of transgenic mice harboringhuman Ig loci for production of human antibodies, and the screening ofsynthetic antibody libraries, such as those generated by mutagenesis ofan antibody variable domain.

EXAMPLE 7 Expression of IFN-L Polypeptide in Transgenic Mice

[0399] To assess the biological activity of IFN-L polypeptide, aconstruct encoding an IFN-L polypeptide/Fc fusion protein under thecontrol of a liver specific ApoE promoter is prepared. The delivery ofthis construct is expected to cause pathological changes that areinformative as to the function of IFN-L polypeptide. Similarly, aconstruct containing the full-length IFN-L polypeptide under the controlof the beta actin promoter is prepared. The delivery of this constructis expected to result in ubiquitous expression.

[0400] To generate these constructs, PCR is used to amplify template DNAsequences encoding an IFN-L polypeptide using primers that correspond tothe 5′ and 3′ ends of the desired sequence and which incorporaterestriction enzyme sites to permit insertion of the amplified productinto an expression vector.

[0401] Following amplification, PCR products are gel purified, digestedwith the appropriate restriction enzymes, and ligated into an expressionvector using standard recombinant DNA techniques. For example, amplifiedIFN-L polypeptide sequences can be cloned into an expression vectorunder the control of the human β-actin promoter as described by Grahamet al., 1997, Nature Genetics, 17:272-74 and Ray et al., 1991, GenesDev. 5:2265-73.

[0402] Following ligation, reaction mixtures are used to transform an E.coli host strain by electroporation and transformants are selected fordrug resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of an appropriateinsert and absence of mutation. The IFN-L polypeptide expression vectoris purified through two rounds of CsCl density gradient centrifugation,cleaved with a suitable restriction enzyme, and the linearized fragmentcontaining the IFN-L polypeptide transgene is purified by gelelectrophoresis. The purified fragment is resuspended in 5 mM Tris, pH7.4, and 0.2 mM EDTA at a concentration of 2 mg/mL.

[0403] Single-cell embryos from BDF1X BDF1 bred mice are injected asdescribed (PCT Pub. No. WO 97/23614). Embryos are cultured overnight ina CO₂ incubator and 15-20 two-cell embryos are transferred to theoviducts of a pseudopregnant CD1 female mice. Offspring obtained fromthe implantation of microinjected embryos are screened by PCRamplification of the integrated transgene in genomic DNA samples asfollows. Ear pieces are digested in 20 mL ear buffer (20 mM Tris, pH8.0, 10 mM EDTA, 0.5% SDS, and 500 mg/mL proteinase K) at 55° C.overnight. The sample is then diluted with 200 mL of TE, and 2 mL of theear sample is used in a PCR reaction using appropriate primers.

[0404] At 8 weeks of age, transgenic founder animals and control animalsare sacrificed for necropsy and pathological analysis. Portions ofspleen are removed and total cellular RNA isolated from the spleensusing the Total RNA Extraction Kit (Qiagen) and transgene expressiondetermined by RT-PCR. RNA recovered from spleens is converted to cDNAusing the SuperScript™ Preamplification System (Gibco-BRL) as follows. Asuitable primer, located in the expression vector sequence and 3′ to theIFN-L polypeptide transgene, is used to prime cDNA synthesis from thetransgene transcripts. Ten mg of total spleen RNA from transgenicfounders and controls is incubated with 1 mM of primer for 10 minutes at70° C. and placed on ice. The reaction is then supplemented with 10 mMTris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 10 mM of each dNTP, 0.1 mMDTT, and 200 U of SuperScript II reverse transcriptase. Followingincubation for 50 minutes at 42° C., the reaction is stopped by heatingfor 15 minutes at 72° C. and digested with 2 U of RNase H for 20 minutesat 37° C. Samples are then amplified by PCR using primers specific forIFN-L polypeptide.

EXAMPLE 8 Biological Activity of IFN-L Polypeptide in Transgenic Mice

[0405] Prior to euthanasia, transgenic animals are weighed, anesthetizedby isofluorane and blood drawn by cardiac puncture. The samples aresubjected to hematology and serum chemistry analysis. Radiography isperformed after terminal exsanguination. Upon gross dissection, majorvisceral organs are subject to weight analysis.

[0406] Following gross dissection, tissues (i.e., liver, spleen,pancreas, stomach, the entire gastrointestinal tract, kidney,reproductive organs, skin and mammary glands, bone, brain, heart, lung,thymus, trachea, esophagus, thyroid, adrenals, urinary bladder, lymphnodes and skeletal muscle) are removed and fixed in 10% bufferedZn-Formalin for histological examination. After fixation, the tissuesare processed into paraffin blocks, and 3 mm sections are obtained. Allsections are stained with hematoxylin and exosin, and are then subjectedto histological analysis.

[0407] The spleen, lymph node, and Peyer's patches of both thetransgenic and the control mice are subjected to immunohistologyanalysis with B cell and T cell specific antibodies as follows. Theformalin fixed paraffin embedded sections are deparaffinized andhydrated in deionized water. The sections are quenched with 3% hydrogenperoxide, blocked with Protein Block (Lipshaw, Pittsburgh, Pa.), andincubated in rat monoclonal anti-mouse B220 and CD3 (Harlan,Indianapolis, Ind.). Antibody binding is detected by biotinylated rabbitanti-rat immunoglobulins and peroxidase conjugated streptavidin(BioGenex, San Ramon, Calif.) with DAB as a chromagen (BioTek, SantaBarbara, Calif.). Sections are counterstained with hematoxylin.

[0408] After necropsy, MLN and sections of spleen and thymus fromtransgenic animals and control littermates are removed. Single cellsuspensions are prepared by gently grinding the tissues with the flatend of a syringe against the bottom of a 100 mm nylon cell strainer(Becton Dickinson, Franklin Lakes, N.J.). Cells are washed twice,counted, and approximately 1×10⁶ cells from each tissue are thenincubated for 10 minutes with 0.5 μg CD16/32(FcγIII/II) Fc block in a 20μL volume. Samples are then stained for 30 minutes at 2-8° C. in a 100μL volume of PBS (lacking Ca⁺ and Mg⁺), 0.1% bovine serum albumin, and0.01% sodium azide with 0.5 μg antibody of FITC or PE-conjugatedmonoclonal antibodies against CD90.2 (Thy-1.2), CD45R (B220),CD11b(Mac-1), Gr-1, CD4, or CD8 (PharMingen, San Diego, Calif.).Following antibody binding, the cells are washed and then analyzed byflow cytometry on a FACScan (Becton Dickinson).

[0409] While the present invention has been described in terms of thepreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationsthat come within the scope of the invention as claimed.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210> SEQ ID NO 1<211> LENGTH: 913 <212> TYPE: DNA <213> ORGANISM: Rattus norvegicus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (53)..(625) <221>NAME/KEY: sig_peptide <222> LOCATION: (53)..(115) <400> SEQUENCE: 1gggtgttgta gatatttttc ctttggaaga aatactgagc accaaggctg ag atg aca 58 MetThr 1 ctg aag tat tta tgg ctg gtg gcc ctc gtg gct cta tac att tca ccc106 Leu Lys Tyr Leu Trp Leu Val Ala Leu Val Ala Leu Tyr Ile Ser Pro 5 1015 atc cag tct cag aac tgt gtg tat ctg gat cat acc atc ttg gaa aac 154Ile Gln Ser Gln Asn Cys Val Tyr Leu Asp His Thr Ile Leu Glu Asn 20 25 30atg aaa ctt ctg agc agc atc agg acc acc ttt ccc tta aga tgt cta 202 MetLys Leu Leu Ser Ser Ile Arg Thr Thr Phe Pro Leu Arg Cys Leu 35 40 45 50aaa gat atc acg gat ttt gag ttt cct caa gag att ctg ctg tac gtc 250 LysAsp Ile Thr Asp Phe Glu Phe Pro Gln Glu Ile Leu Leu Tyr Val 55 60 65 cagcat gtg aaa aag gac ata aag gca gtc acc tat cat ata tct tct 298 Gln HisVal Lys Lys Asp Ile Lys Ala Val Thr Tyr His Ile Ser Ser 70 75 80 ctg gcgcta att att ttc agt ctt aaa gac tcc atc tcc ctg gcg aca 346 Leu Ala LeuIle Ile Phe Ser Leu Lys Asp Ser Ile Ser Leu Ala Thr 85 90 95 gag gaa cgcttg gaa cgt atc aga tcg gga ctt ttc aaa caa gtg cag 394 Glu Glu Arg LeuGlu Arg Ile Arg Ser Gly Leu Phe Lys Gln Val Gln 100 105 110 caa gct cgagag tgc atg gta gac gag gag aac aag aac acg gag gag 442 Gln Ala Arg GluCys Met Val Asp Glu Glu Asn Lys Asn Thr Glu Glu 115 120 125 130 gac agtaca tca caa cat cct cac tca gag ggc ttc aag gca gtc tac 490 Asp Ser ThrSer Gln His Pro His Ser Glu Gly Phe Lys Ala Val Tyr 135 140 145 ctg gaattg aac aag tat ttc ttc aga atc aga aag ttc ctg gta aat 538 Leu Glu LeuAsn Lys Tyr Phe Phe Arg Ile Arg Lys Phe Leu Val Asn 150 155 160 aag aaatac agt ttc tgt gcc tgg aag att gtc gtg gtg gaa ata aga 586 Lys Lys TyrSer Phe Cys Ala Trp Lys Ile Val Val Val Glu Ile Arg 165 170 175 aga tgtttc agt ata ttt tac aaa cta ctc aac atg aat tgagaatcat 635 Arg Cys PheSer Ile Phe Tyr Lys Leu Leu Asn Met Asn 180 185 190 ccagcttcaagcaagaactt agatagaagt tgtgactgct caaatgtccc caagaacgct 695 tgattctaaggctattgcga gtctgctgct acacacttcg gacgcaagac ttttcaaggt 755 cagggttcaaggtagtacag tcaaaggaag tcttatgtta agcaaaagaa aaatttcagt 815 ggaaaagctagcagaaatgt caacttgtca aaaaaacaac ttatggatta tggcattgac 875 gttactagcaaaaaaaataa aacaaaaaaa aacaaaaa 913 <210> SEQ ID NO 2 <211> LENGTH: 191<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 2 MetThr Leu Lys Tyr Leu Trp Leu Val Ala Leu Val Ala Leu Tyr Ile 1 5 10 15Ser Pro Ile Gln Ser Gln Asn Cys Val Tyr Leu Asp His Thr Ile Leu 20 25 30Glu Asn Met Lys Leu Leu Ser Ser Ile Arg Thr Thr Phe Pro Leu Arg 35 40 45Cys Leu Lys Asp Ile Thr Asp Phe Glu Phe Pro Gln Glu Ile Leu Leu 50 55 60Tyr Val Gln His Val Lys Lys Asp Ile Lys Ala Val Thr Tyr His Ile 65 70 7580 Ser Ser Leu Ala Leu Ile Ile Phe Ser Leu Lys Asp Ser Ile Ser Leu 85 9095 Ala Thr Glu Glu Arg Leu Glu Arg Ile Arg Ser Gly Leu Phe Lys Gln 100105 110 Val Gln Gln Ala Arg Glu Cys Met Val Asp Glu Glu Asn Lys Asn Thr115 120 125 Glu Glu Asp Ser Thr Ser Gln His Pro His Ser Glu Gly Phe LysAla 130 135 140 Val Tyr Leu Glu Leu Asn Lys Tyr Phe Phe Arg Ile Arg LysPhe Leu 145 150 155 160 Val Asn Lys Lys Tyr Ser Phe Cys Ala Trp Lys IleVal Val Val Glu 165 170 175 Ile Arg Arg Cys Phe Ser Ile Phe Tyr Lys LeuLeu Asn Met Asn 180 185 190 <210> SEQ ID NO 3 <211> LENGTH: 168 <212>TYPE: PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 3 Cys ValTyr Leu Asp His Thr Ile Leu Glu Asn Met Lys Leu Leu Ser 1 5 10 15 SerIle Arg Thr Thr Phe Pro Leu Arg Cys Leu Lys Asp Ile Thr Asp 20 25 30 PheGlu Phe Pro Gln Glu Ile Leu Leu Tyr Val Gln His Val Lys Lys 35 40 45 AspIle Lys Ala Val Thr Tyr His Ile Ser Ser Leu Ala Leu Ile Ile 50 55 60 PheSer Leu Lys Asp Ser Ile Ser Leu Ala Thr Glu Glu Arg Leu Glu 65 70 75 80Arg Ile Arg Ser Gly Leu Phe Lys Gln Val Gln Gln Ala Arg Glu Cys 85 90 95Met Val Asp Glu Glu Asn Lys Asn Thr Glu Glu Asp Ser Thr Ser Gln 100 105110 His Pro His Ser Glu Gly Phe Lys Ala Val Tyr Leu Glu Leu Asn Lys 115120 125 Tyr Phe Phe Arg Ile Arg Lys Phe Leu Val Asn Lys Lys Tyr Ser Phe130 135 140 Cys Ala Trp Lys Ile Val Val Val Glu Ile Arg Arg Cys Phe SerIle 145 150 155 160 Phe Tyr Lys Leu Leu Asn Met Asn 165 <210> SEQ ID NO4 <211> LENGTH: 1836 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (575)..(1195) <221>NAME/KEY: sig_peptide <222> LOCATION: (575)..(655) <400> SEQUENCE: 4aagcttaatt taacaaaatt ggaaaaacct aaactatact gtgctctggt gacctagcaa 60tcaaataatc acagtcattt ggtcaatgtc tatgattaac tcaatgagac aggatgtttg 120gctatagcac caggtacaaa aaatatattt tcatgaagga tcactccctc ttatgtaata 180gatttgggtg agtgagtgag tgagtgagtg catggactca cagcttttgg ctttctgaaa 240taccctgcat cagtcttgtt atgatgattc cttagtgctg ggatggatca tccaggcatt 300taaggtaaca cgatggtaat tctttgctca tttttcaggg aaaaaaaaaa gttatcactt 360ccaaagtcgg catagtcacc cgaagtaaaa aaaaaaaaaa aaaaaaaaag cctcagaggc 420aaaggaaagg ggccgcaacc ttggttaact gtgaaatgac gaatgagaaa actcctcctg 480ctgaagatat tcaggtatat aaaggcacat gaaggaaaac tcaaaacatc attgtcatat 540acacatcttc tggatttttt agcttgcaaa aaaa atg agc acc aaa cct gat atg 595Met Ser Thr Lys Pro Asp Met 1 5 att caa aag tgt ttg tgg ctt gag atc cttatg ggt ata ttc att gct 643 Ile Gln Lys Cys Leu Trp Leu Glu Ile Leu MetGly Ile Phe Ile Ala 10 15 20 ggc acc cta tcc ctg gac tgt aac tta ctg aacgtt cac ctg aga aga 691 Gly Thr Leu Ser Leu Asp Cys Asn Leu Leu Asn ValHis Leu Arg Arg 25 30 35 gtc acc tgg caa aat ctg aga cat ctg agt agt atgagc aat tca ttt 739 Val Thr Trp Gln Asn Leu Arg His Leu Ser Ser Met SerAsn Ser Phe 40 45 50 55 cct gta gaa tgt cta cga gaa aac ata gct ttt gagttg ccc caa gag 787 Pro Val Glu Cys Leu Arg Glu Asn Ile Ala Phe Glu LeuPro Gln Glu 60 65 70 ttt ctg caa tac acc caa cct atg aag agg gac atc aagaag gcc ttc 835 Phe Leu Gln Tyr Thr Gln Pro Met Lys Arg Asp Ile Lys LysAla Phe 75 80 85 tat gaa atg tcc cta cag gcc ttc aac atc ttc agc caa cacacc ttc 883 Tyr Glu Met Ser Leu Gln Ala Phe Asn Ile Phe Ser Gln His ThrPhe 90 95 100 aaa tat tgg aaa gag aga cac ctc aaa caa atc caa ata ggactt gat 931 Lys Tyr Trp Lys Glu Arg His Leu Lys Gln Ile Gln Ile Gly LeuAsp 105 110 115 cag caa gca gag tac ctg aac caa tgc ttg gag gaa gac gagaat gaa 979 Gln Gln Ala Glu Tyr Leu Asn Gln Cys Leu Glu Glu Asp Glu AsnGlu 120 125 130 135 aat gaa gac atg aaa gaa atg aaa gag aat gag atg aaaccc tca gaa 1027 Asn Glu Asp Met Lys Glu Met Lys Glu Asn Glu Met Lys ProSer Glu 140 145 150 gcc agg gtc ccc cag ctg agc agc ctg gaa ctg agg agatat ttc cac 1075 Ala Arg Val Pro Gln Leu Ser Ser Leu Glu Leu Arg Arg TyrPhe His 155 160 165 agg ata gac aat ttc ctg aaa gaa aag aaa tac agt gactgt gcc tgg 1123 Arg Ile Asp Asn Phe Leu Lys Glu Lys Lys Tyr Ser Asp CysAla Trp 170 175 180 gag att gtc cga gtg gaa atc aga aga tgt ttg tat tacttt tac aaa 1171 Glu Ile Val Arg Val Glu Ile Arg Arg Cys Leu Tyr Tyr PheTyr Lys 185 190 195 ttt aca gct cta ttc agg agg aaa taaggtatatttttggaatt aaaattcctt 1225 Phe Thr Ala Leu Phe Arg Arg Lys 200 205ttccctccga aatctctttc tccttctcct cctccatctt ctttttaagg attgttgtgc 1285tgtcctgtaa gcctgtcctc agttggactg gtagcctcgg aacatcaggg acactcacct 1345ctctaaggag aggtaatgcc aaccatcctc agggtgacca agagtctcct tagaaagtct 1405ttaagacatt tttaaaggaa taagattccc tctccgtctt cttctattct ctcttgctct 1465tttctgtggc cattttgaaa gagctttgct atatatacca cctgtggact tcaccaagac 1525aatggctaga ggatagggag cagagaatgt tgcaaaatgg taacatttca atgacttaac 1585tgttttgctg ccaaggttgc ttatcctatg aaaattcagc acattaaaag agcttataca 1645tgctccctag agtcaatact cttgcatttt ccccctcctg ctcgggggga aaaaggttga 1705catttctggc ccatttcctt ctcagcttgg tttgtttgaa ttgatgcttg tggaatggta 1765tttcattact ttaagagtga agatccatag tgaaattgga tggatggttg aattagacga 1825ccattaagct t 1836 <210> SEQ ID NO 5 <211> LENGTH: 207 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Ser Thr Lys Pro AspMet Ile Gln Lys Cys Leu Trp Leu Glu Ile 1 5 10 15 Leu Met Gly Ile PheIle Ala Gly Thr Leu Ser Leu Asp Cys Asn Leu 20 25 30 Leu Asn Val His LeuArg Arg Val Thr Trp Gln Asn Leu Arg His Leu 35 40 45 Ser Ser Met Ser AsnSer Phe Pro Val Glu Cys Leu Arg Glu Asn Ile 50 55 60 Ala Phe Glu Leu ProGln Glu Phe Leu Gln Tyr Thr Gln Pro Met Lys 65 70 75 80 Arg Asp Ile LysLys Ala Phe Tyr Glu Met Ser Leu Gln Ala Phe Asn 85 90 95 Ile Phe Ser GlnHis Thr Phe Lys Tyr Trp Lys Glu Arg His Leu Lys 100 105 110 Gln Ile GlnIle Gly Leu Asp Gln Gln Ala Glu Tyr Leu Asn Gln Cys 115 120 125 Leu GluGlu Asp Glu Asn Glu Asn Glu Asp Met Lys Glu Met Lys Glu 130 135 140 AsnGlu Met Lys Pro Ser Glu Ala Arg Val Pro Gln Leu Ser Ser Leu 145 150 155160 Glu Leu Arg Arg Tyr Phe His Arg Ile Asp Asn Phe Leu Lys Glu Lys 165170 175 Lys Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg Val Glu Ile Arg Arg180 185 190 Cys Leu Tyr Tyr Phe Tyr Lys Phe Thr Ala Leu Phe Arg Arg Lys195 200 205 <210> SEQ ID NO 6 <211> LENGTH: 178 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 6 Cys Asn Leu Leu Asn Val His LeuArg Arg Val Thr Trp Gln Asn Leu 1 5 10 15 Arg His Leu Ser Ser Met SerAsn Ser Phe Pro Val Glu Cys Leu Arg 20 25 30 Glu Asn Ile Ala Phe Glu LeuPro Gln Glu Phe Leu Gln Tyr Thr Gln 35 40 45 Pro Met Lys Arg Asp Ile LysLys Ala Phe Tyr Glu Met Ser Leu Gln 50 55 60 Ala Phe Asn Ile Phe Ser GlnHis Thr Phe Lys Tyr Trp Lys Glu Arg 65 70 75 80 His Leu Lys Gln Ile GlnIle Gly Leu Asp Gln Gln Ala Glu Tyr Leu 85 90 95 Asn Gln Cys Leu Glu GluAsp Glu Asn Glu Asn Glu Asp Met Lys Glu 100 105 110 Met Lys Glu Asn GluMet Lys Pro Ser Glu Ala Arg Val Pro Gln Leu 115 120 125 Ser Ser Leu GluLeu Arg Arg Tyr Phe His Arg Ile Asp Asn Phe Leu 130 135 140 Lys Glu LysLys Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg Val Glu 145 150 155 160 IleArg Arg Cys Leu Tyr Tyr Phe Tyr Lys Phe Thr Ala Leu Phe Arg 165 170 175Arg Lys <210> SEQ ID NO 7 <211> LENGTH: 187 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 7 Met Thr Asn Lys Cys Ile Leu GlnIle Ala Leu Leu Leu Cys His Ser 1 5 10 15 Thr Thr Ala Leu Ser Met SerTyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys GlnLys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys AspArg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln PheGln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln AsnIle Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn GluThr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile AsnHis Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp PheThr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg Tyr TyrGly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 HisCys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175Phe Ile Asn Lys Leu Thr Gly Tyr Leu Arg Asn 180 185 <210> SEQ ID NO 8<211> LENGTH: 520 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Rat IFN- like polypeptide cDNA insert and partial pAMG21vector sequence <221> NAME/KEY: CDS <222> LOCATION: (4)..(510) <400>SEQUENCE: 8 cat atg tgt gta tat ctc gat cat act atc ttg gag aat atg aaactt 48 Met Cys Val Tyr Leu Asp His Thr Ile Leu Glu Asn Met Lys Leu 1 510 15 ctg agc agc atc cgt acc acc ttt cct ctg cgt tgt ctg aaa gat atc 96Leu Ser Ser Ile Arg Thr Thr Phe Pro Leu Arg Cys Leu Lys Asp Ile 20 25 30acg gat ttt gag ttt cct caa gag att ctg ctg tac gtc cag cat gtg 144 ThrAsp Phe Glu Phe Pro Gln Glu Ile Leu Leu Tyr Val Gln His Val 35 40 45 aaaaag gac ata aag gca gtc acc tat cat ata tct tct ctg gcg cta 192 Lys LysAsp Ile Lys Ala Val Thr Tyr His Ile Ser Ser Leu Ala Leu 50 55 60 att attttc agt ctt aaa gac tcc atc tcc ctg gcg aca gag gaa cgc 240 Ile Ile PheSer Leu Lys Asp Ser Ile Ser Leu Ala Thr Glu Glu Arg 65 70 75 ttg gaa cgtatc aga tcg gga ctt ttc aaa caa gtg cag caa gct cga 288 Leu Glu Arg IleArg Ser Gly Leu Phe Lys Gln Val Gln Gln Ala Arg 80 85 90 95 gag tgc atggta gac gag gag aac aag aac acg gag gag gac agt aca 336 Glu Cys Met ValAsp Glu Glu Asn Lys Asn Thr Glu Glu Asp Ser Thr 100 105 110 tca caa catcct cac tca gag ggc ttc aag gca gtc tac ctg gaa ttg 384 Ser Gln His ProHis Ser Glu Gly Phe Lys Ala Val Tyr Leu Glu Leu 115 120 125 aac aag tatttc ttc aga atc aga aag ttc ctg gta aat aag aaa tac 432 Asn Lys Tyr PhePhe Arg Ile Arg Lys Phe Leu Val Asn Lys Lys Tyr 130 135 140 agt ttc tgtgcc tgg aag att gtc gtg gtg gaa att cgt cgt tgt ttc 480 Ser Phe Cys AlaTrp Lys Ile Val Val Val Glu Ile Arg Arg Cys Phe 145 150 155 agt att ttttac aaa ctg ctg aac atg aat taatggatcc 520 Ser Ile Phe Tyr Lys Leu LeuAsn Met Asn 160 165 <210> SEQ ID NO 9 <211> LENGTH: 169 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Rat IFN- likepolypeptide cDNA insert and partial pAMG21 vector sequence <400>SEQUENCE: 9 Met Cys Val Tyr Leu Asp His Thr Ile Leu Glu Asn Met Lys LeuLeu 1 5 10 15 Ser Ser Ile Arg Thr Thr Phe Pro Leu Arg Cys Leu Lys AspIle Thr 20 25 30 Asp Phe Glu Phe Pro Gln Glu Ile Leu Leu Tyr Val Gln HisVal Lys 35 40 45 Lys Asp Ile Lys Ala Val Thr Tyr His Ile Ser Ser Leu AlaLeu Ile 50 55 60 Ile Phe Ser Leu Lys Asp Ser Ile Ser Leu Ala Thr Glu GluArg Leu 65 70 75 80 Glu Arg Ile Arg Ser Gly Leu Phe Lys Gln Val Gln GlnAla Arg Glu 85 90 95 Cys Met Val Asp Glu Glu Asn Lys Asn Thr Glu Glu AspSer Thr Ser 100 105 110 Gln His Pro His Ser Glu Gly Phe Lys Ala Val TyrLeu Glu Leu Asn 115 120 125 Lys Tyr Phe Phe Arg Ile Arg Lys Phe Leu ValAsn Lys Lys Tyr Ser 130 135 140 Phe Cys Ala Trp Lys Ile Val Val Val GluIle Arg Arg Cys Phe Ser 145 150 155 160 Ile Phe Tyr Lys Leu Leu Asn MetAsn 165 <210> SEQ ID NO 10 <211> LENGTH: 520 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Rat IFN- like polypeptide cDNAinsert and partial pAMG21 vector sequence <221> NAME/KEY: CDS <222>LOCATION: (4)..(510) <400> SEQUENCE: 10 cat atg tgt gta tat ctc gat catact atc ttg gag aat atg aaa ctt 48 Met Cys Val Tyr Leu Asp His Thr IleLeu Glu Asn Met Lys Leu 1 5 10 15 ctg agc agc atc cgt acc acc ttt cctctg cgt tgt ctg aaa gat atc 96 Leu Ser Ser Ile Arg Thr Thr Phe Pro LeuArg Cys Leu Lys Asp Ile 20 25 30 acg gat ttt gag ttt cct caa gag att ctgctg tac gtc cag cat gtg 144 Thr Asp Phe Glu Phe Pro Gln Glu Ile Leu LeuTyr Val Gln His Val 35 40 45 aaa aag gac atc aag gca gtc acc tat cat atctct tct ctg gcg ctg 192 Lys Lys Asp Ile Lys Ala Val Thr Tyr His Ile SerSer Leu Ala Leu 50 55 60 att att ttc agt ctt aaa gac tcc atc tcc ctg gcgaca gag gaa cgc 240 Ile Ile Phe Ser Leu Lys Asp Ser Ile Ser Leu Ala ThrGlu Glu Arg 65 70 75 ttg gaa cgt atc cgt tct ggt ctt ttc aaa caa gtg cagcaa gct cgt 288 Leu Glu Arg Ile Arg Ser Gly Leu Phe Lys Gln Val Gln GlnAla Arg 80 85 90 95 gag tgc atg gta gac gag gag aac aag aac acg gag gaggac agt aca 336 Glu Cys Met Val Asp Glu Glu Asn Lys Asn Thr Glu Glu AspSer Thr 100 105 110 tca caa cat cct cac tca gag ggc ttc aag gca gtc tacctg gaa ttg 384 Ser Gln His Pro His Ser Glu Gly Phe Lys Ala Val Tyr LeuGlu Leu 115 120 125 aac aag tat ttc ttc cgt atc cgt aag ttc ctg gta aataag aaa tac 432 Asn Lys Tyr Phe Phe Arg Ile Arg Lys Phe Leu Val Asn LysLys Tyr 130 135 140 agt ttc tgt gcc tgg aag att gtc gtg gtg gaa att cgtcgt tct ttc 480 Ser Phe Cys Ala Trp Lys Ile Val Val Val Glu Ile Arg ArgSer Phe 145 150 155 agt att ttt tac aaa ctg ctg aac atg aat taatggatcc520 Ser Ile Phe Tyr Lys Leu Leu Asn Met Asn 160 165 <210> SEQ ID NO 11<211> LENGTH: 169 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Rat IFN- like polypeptide cDNA insert and partial pAMG21vector sequence <400> SEQUENCE: 11 Met Cys Val Tyr Leu Asp His Thr IleLeu Glu Asn Met Lys Leu Leu 1 5 10 15 Ser Ser Ile Arg Thr Thr Phe ProLeu Arg Cys Leu Lys Asp Ile Thr 20 25 30 Asp Phe Glu Phe Pro Gln Glu IleLeu Leu Tyr Val Gln His Val Lys 35 40 45 Lys Asp Ile Lys Ala Val Thr TyrHis Ile Ser Ser Leu Ala Leu Ile 50 55 60 Ile Phe Ser Leu Lys Asp Ser IleSer Leu Ala Thr Glu Glu Arg Leu 65 70 75 80 Glu Arg Ile Arg Ser Gly LeuPhe Lys Gln Val Gln Gln Ala Arg Glu 85 90 95 Cys Met Val Asp Glu Glu AsnLys Asn Thr Glu Glu Asp Ser Thr Ser 100 105 110 Gln His Pro His Ser GluGly Phe Lys Ala Val Tyr Leu Glu Leu Asn 115 120 125 Lys Tyr Phe Phe ArgIle Arg Lys Phe Leu Val Asn Lys Lys Tyr Ser 130 135 140 Phe Cys Ala TrpLys Ile Val Val Val Glu Ile Arg Arg Ser Phe Ser 145 150 155 160 Ile PheTyr Lys Leu Leu Asn Met Asn 165 <210> SEQ ID NO 12 <211> LENGTH: 568<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Human IFN- likepolypeptide cDNA insert and partial pAMG21 vector sequence <221>NAME/KEY: CDS <222> LOCATION: (22)..(558) <400> SEQUENCE: 12 tctagaaaggaggaataaca t atg tgt aac ctg ctg aac gtt cac ctg cgt 51 Met Cys Asn LeuLeu Asn Val His Leu Arg 1 5 10 cgt gtt acc tgg caa aat ctg aga cat ctgagt agt atg agc aat tca 99 Arg Val Thr Trp Gln Asn Leu Arg His Leu SerSer Met Ser Asn Ser 15 20 25 ttt cct gta gaa tgt cta cga gaa aac ata gctttt gag ttg ccc caa 147 Phe Pro Val Glu Cys Leu Arg Glu Asn Ile Ala PheGlu Leu Pro Gln 30 35 40 gag ttt ctg caa tac acc caa cct atg aag agg gacatc aag aag gcc 195 Glu Phe Leu Gln Tyr Thr Gln Pro Met Lys Arg Asp IleLys Lys Ala 45 50 55 ttc tat gaa atg tcc cta cag gcc ttc aac atc ttc agccaa cac acc 243 Phe Tyr Glu Met Ser Leu Gln Ala Phe Asn Ile Phe Ser GlnHis Thr 60 65 70 ttc aaa tat tgg aaa gag aga cac ctc aaa caa atc caa atagga ctt 291 Phe Lys Tyr Trp Lys Glu Arg His Leu Lys Gln Ile Gln Ile GlyLeu 75 80 85 90 gat cag caa gca gag tac ctg aac caa tgc ttg gag gaa gacgag aat 339 Asp Gln Gln Ala Glu Tyr Leu Asn Gln Cys Leu Glu Glu Asp GluAsn 95 100 105 gaa aat gaa gac atg aaa gaa atg aaa gag aat gag atg aaaccc tca 387 Glu Asn Glu Asp Met Lys Glu Met Lys Glu Asn Glu Met Lys ProSer 110 115 120 gaa gcc agg gtc ccc cag ctg agc agc ctg gaa ctg agg agatat ttc 435 Glu Ala Arg Val Pro Gln Leu Ser Ser Leu Glu Leu Arg Arg TyrPhe 125 130 135 cac agg ata gac aat ttc ctg aaa gaa aag aaa tac agt gactgt gcc 483 His Arg Ile Asp Asn Phe Leu Lys Glu Lys Lys Tyr Ser Asp CysAla 140 145 150 tgg gag att gtc cga gtg gaa atc cgt cgt tgc ctg tac tacttt tac 531 Trp Glu Ile Val Arg Val Glu Ile Arg Arg Cys Leu Tyr Tyr PheTyr 155 160 165 170 aaa ttt acc gct ctg ttc cgt cgt aaa taatggatcc 568Lys Phe Thr Ala Leu Phe Arg Arg Lys 175 <210> SEQ ID NO 13 <211> LENGTH:179 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Rat IFN-like polypeptide cDNA insert and partial pAMG21 vector sequence <400>SEQUENCE: 13 Met Cys Asn Leu Leu Asn Val His Leu Arg Arg Val Thr Trp GlnAsn 1 5 10 15 Leu Arg His Leu Ser Ser Met Ser Asn Ser Phe Pro Val GluCys Leu 20 25 30 Arg Glu Asn Ile Ala Phe Glu Leu Pro Gln Glu Phe Leu GlnTyr Thr 35 40 45 Gln Pro Met Lys Arg Asp Ile Lys Lys Ala Phe Tyr Glu MetSer Leu 50 55 60 Gln Ala Phe Asn Ile Phe Ser Gln His Thr Phe Lys Tyr TrpLys Glu 65 70 75 80 Arg His Leu Lys Gln Ile Gln Ile Gly Leu Asp Gln GlnAla Glu Tyr 85 90 95 Leu Asn Gln Cys Leu Glu Glu Asp Glu Asn Glu Asn GluAsp Met Lys 100 105 110 Glu Met Lys Glu Asn Glu Met Lys Pro Ser Glu AlaArg Val Pro Gln 115 120 125 Leu Ser Ser Leu Glu Leu Arg Arg Tyr Phe HisArg Ile Asp Asn Phe 130 135 140 Leu Lys Glu Lys Lys Tyr Ser Asp Cys AlaTrp Glu Ile Val Arg Val 145 150 155 160 Glu Ile Arg Arg Cys Leu Tyr TyrPhe Tyr Lys Phe Thr Ala Leu Phe 165 170 175 Arg Arg Lys <210> SEQ ID NO14 <211> LENGTH: 568 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Human IFN- like polypeptide cDNA insert and partial pAMG21vector sequence <221> NAME/KEY: CDS <222> LOCATION: (22)..(558) <400>SEQUENCE: 14 tctagaaagg aggaataaca t atg tgt aac ctg ctg aac gtt cac ctgcgt 51 Met Cys Asn Leu Leu Asn Val His Leu Arg 1 5 10 cgt gtt acc tggcaa aat ctg aga cat ctg agt agt atg agc aat tca 99 Arg Val Thr Trp GlnAsn Leu Arg His Leu Ser Ser Met Ser Asn Ser 15 20 25 ttt cct gta gaa tgtcta cga gaa aac ata gct ttt gag ttg ccc caa 147 Phe Pro Val Glu Cys LeuArg Glu Asn Ile Ala Phe Glu Leu Pro Gln 30 35 40 gag ttc ctg caa tac acccaa cct atg aag agg gac atc aag aag gcc 195 Glu Phe Leu Gln Tyr Thr GlnPro Met Lys Arg Asp Ile Lys Lys Ala 45 50 55 ttc tat gaa atg tcc cta caggcc ttc aac atc ttc agc caa cac acc 243 Phe Tyr Glu Met Ser Leu Gln AlaPhe Asn Ile Phe Ser Gln His Thr 60 65 70 ttc aaa tat tgg aaa gag aga cacctc aaa caa atc caa ata gga ctt 291 Phe Lys Tyr Trp Lys Glu Arg His LeuLys Gln Ile Gln Ile Gly Leu 75 80 85 90 gat cag caa gca gag tac ctg aaccaa tgc ttg gag gaa gac gag aat 339 Asp Gln Gln Ala Glu Tyr Leu Asn GlnCys Leu Glu Glu Asp Glu Asn 95 100 105 gaa aat gaa gac atg aaa gaa atgaaa gag aat gag atg aaa ccc tca 387 Glu Asn Glu Asp Met Lys Glu Met LysGlu Asn Glu Met Lys Pro Ser 110 115 120 gaa gcc agg gtc ccc cag ctg agcagc ctg gaa ctg agg aga tat ttc 435 Glu Ala Arg Val Pro Gln Leu Ser SerLeu Glu Leu Arg Arg Tyr Phe 125 130 135 cac agg ata gac aat ttc ctg aaagaa aag aaa tac agt gac tgt gcc 483 His Arg Ile Asp Asn Phe Leu Lys GluLys Lys Tyr Ser Asp Cys Ala 140 145 150 tgg gag att gtc cga gtg gaa atccgt cgt tct ctg tac tac ttt tac 531 Trp Glu Ile Val Arg Val Glu Ile ArgArg Ser Leu Tyr Tyr Phe Tyr 155 160 165 170 aaa ttt acc gct ctg ttc cgtcgt aaa taatggatcc 568 Lys Phe Thr Ala Leu Phe Arg Arg Lys 175 <210> SEQID NO 15 <211> LENGTH: 179 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Human IFN- like polypeptide cDNA insert and partialpAMG21 vector sequence <400> SEQUENCE: 15 Met Cys Asn Leu Leu Asn ValHis Leu Arg Arg Val Thr Trp Gln Asn 1 5 10 15 Leu Arg His Leu Ser SerMet Ser Asn Ser Phe Pro Val Glu Cys Leu 20 25 30 Arg Glu Asn Ile Ala PheGlu Leu Pro Gln Glu Phe Leu Gln Tyr Thr 35 40 45 Gln Pro Met Lys Arg AspIle Lys Lys Ala Phe Tyr Glu Met Ser Leu 50 55 60 Gln Ala Phe Asn Ile PheSer Gln His Thr Phe Lys Tyr Trp Lys Glu 65 70 75 80 Arg His Leu Lys GlnIle Gln Ile Gly Leu Asp Gln Gln Ala Glu Tyr 85 90 95 Leu Asn Gln Cys LeuGlu Glu Asp Glu Asn Glu Asn Glu Asp Met Lys 100 105 110 Glu Met Lys GluAsn Glu Met Lys Pro Ser Glu Ala Arg Val Pro Gln 115 120 125 Leu Ser SerLeu Glu Leu Arg Arg Tyr Phe His Arg Ile Asp Asn Phe 130 135 140 Leu LysGlu Lys Lys Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg Val 145 150 155 160Glu Ile Arg Arg Ser Leu Tyr Tyr Phe Tyr Lys Phe Thr Ala Leu Phe 165 170175 Arg Arg Lys <210> SEQ ID NO 16 <211> LENGTH: 556 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Human IFN- likepolypeptide cDNA insert and partial pAMG21 vector sequence <221>NAME/KEY: CDS <222> LOCATION: (1)..(546) <400> SEQUENCE: 16 cat atg ctggac tgt aac ctg ctg aac gtt cac ctg cgt cgt gtt acc 48 His Met Leu AspCys Asn Leu Leu Asn Val His Leu Arg Arg Val Thr 1 5 10 15 tgg caa aatctg aga cat ctg agt agt atg agc aat tca ttt cct gta 96 Trp Gln Asn LeuArg His Leu Ser Ser Met Ser Asn Ser Phe Pro Val 20 25 30 gaa tgt cta cgagaa aac ata gct ttt gag ttg ccc caa gag ttt ctg 144 Glu Cys Leu Arg GluAsn Ile Ala Phe Glu Leu Pro Gln Glu Phe Leu 35 40 45 caa tac acc caa cctatg aag agg gac atc aag aag gcc ttc tat gaa 192 Gln Tyr Thr Gln Pro MetLys Arg Asp Ile Lys Lys Ala Phe Tyr Glu 50 55 60 atg tcc cta cag gcc ttcaac atc ttc agc caa cac acc ttc aaa tat 240 Met Ser Leu Gln Ala Phe AsnIle Phe Ser Gln His Thr Phe Lys Tyr 65 70 75 80 tgg aaa gag aga cac ctcaaa caa atc caa ata gga ctt gat cag caa 288 Trp Lys Glu Arg His Leu LysGln Ile Gln Ile Gly Leu Asp Gln Gln 85 90 95 gca gag tac ctg aac caa tgcttg gag gaa gac gag aat gaa aat gaa 336 Ala Glu Tyr Leu Asn Gln Cys LeuGlu Glu Asp Glu Asn Glu Asn Glu 100 105 110 gac atg aaa gaa atg aaa gagaat gag atg aaa ccc tca gaa gcc agg 384 Asp Met Lys Glu Met Lys Glu AsnGlu Met Lys Pro Ser Glu Ala Arg 115 120 125 gtc ccc cag ctg agc agc ctggaa ctg agg aga tat ttc cac agg ata 432 Val Pro Gln Leu Ser Ser Leu GluLeu Arg Arg Tyr Phe His Arg Ile 130 135 140 gac aat ttc ctg aaa gaa aagaaa tac agt gac tgt gcc tgg gag att 480 Asp Asn Phe Leu Lys Glu Lys LysTyr Ser Asp Cys Ala Trp Glu Ile 145 150 155 160 gtc cga gtg gaa atc cgtcgt tgc ctg tac tac ttt tac aaa ttt acc 528 Val Arg Val Glu Ile Arg ArgCys Leu Tyr Tyr Phe Tyr Lys Phe Thr 165 170 175 gct ctg ttc cgt cgt aaataatggatcc 556 Ala Leu Phe Arg Arg Lys 180 <210> SEQ ID NO 17 <211>LENGTH: 182 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Human IFN- like polypeptide cDNA insert and partial pAMG21 vectorsequence <400> SEQUENCE: 17 His Met Leu Asp Cys Asn Leu Leu Asn Val HisLeu Arg Arg Val Thr 1 5 10 15 Trp Gln Asn Leu Arg His Leu Ser Ser MetSer Asn Ser Phe Pro Val 20 25 30 Glu Cys Leu Arg Glu Asn Ile Ala Phe GluLeu Pro Gln Glu Phe Leu 35 40 45 Gln Tyr Thr Gln Pro Met Lys Arg Asp IleLys Lys Ala Phe Tyr Glu 50 55 60 Met Ser Leu Gln Ala Phe Asn Ile Phe SerGln His Thr Phe Lys Tyr 65 70 75 80 Trp Lys Glu Arg His Leu Lys Gln IleGln Ile Gly Leu Asp Gln Gln 85 90 95 Ala Glu Tyr Leu Asn Gln Cys Leu GluGlu Asp Glu Asn Glu Asn Glu 100 105 110 Asp Met Lys Glu Met Lys Glu AsnGlu Met Lys Pro Ser Glu Ala Arg 115 120 125 Val Pro Gln Leu Ser Ser LeuGlu Leu Arg Arg Tyr Phe His Arg Ile 130 135 140 Asp Asn Phe Leu Lys GluLys Lys Tyr Ser Asp Cys Ala Trp Glu Ile 145 150 155 160 Val Arg Val GluIle Arg Arg Cys Leu Tyr Tyr Phe Tyr Lys Phe Thr 165 170 175 Ala Leu PheArg Arg Lys 180 <210> SEQ ID NO 18 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: Human immunodeficiency virus type 1 <400> SEQUENCE: 18Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> SEQ ID NO 19<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Internalizing domain derived from HIV tat protein <400>SEQUENCE: 19 Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10 15 <210> SEQ ID NO 20 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Rattus norvegicus <400> SEQUENCE: 20 atgacactga agtatttatg g21 <210> SEQ ID NO 21 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Rattus norvegicus <400> SEQUENCE: 21 attcatgttg agtagtttgt a 21 <210>SEQ ID NO 22 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer 1825-22 <400> SEQUENCE: 22 gaataacatatgtgtgtata tctcgatcat actatcttgg agaatatg 48 <210> SEQ ID NO 23 <211>LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer 1825-21 <400> SEQUENCE: 23 ccgcggatcc attaattcat gttcagcagtttgtaaaaaa tactgaaaca acgacgaatt 60 tcc 63 <210> SEQ ID NO 24 <211>LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer 1909-56 <400> SEQUENCE: 24 ccgcggatcc attaattcat gttcagcagtttgtaaaaaa tactgaaaga acgacgaatt 60 tcc 63 <210> SEQ ID NO 25 <211>LENGTH: 67 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer 1967-32 <400> SEQUENCE: 25 ttgatctaga aaggaggaat aacatatgtgtaacctgctg aacgttcacc tgcgtcgtgt 60 tacctgg 67 <210> SEQ ID NO 26 <211>LENGTH: 71 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer 1982-14 <400> SEQUENCE: 26 ccgcggatcc attatttacg acggaacagagcggtaaatt tgtaaaagta gtacaggcaa 60 cgacgatttc c 71 <210> SEQ ID NO 27<211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: PCR primer 1967-33 <400> SEQUENCE: 27 ccgcggatcc attatttacgacggaacaga gcggtaaatt tgtaaaagta gtacagagaa 60 cgacggattt cc 72 <210>SEQ ID NO 28 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer 2103-87 <400> SEQUENCE: 28 aaggagcatatgctggactg taacctgctg aacgttcac 39 <210> SEQ ID NO 29 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: PCR primer1200-54 <400> SEQUENCE: 29 gttattgctc agcggtggca 20 <210> SEQ ID NO 30<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: PCR primer 1847-77 <400> SEQUENCE: 30 cccaagctta ccatgacactgaagtattta tg 32 <210> SEQ ID NO 31 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: PCR primer 1847-78<400> SEQUENCE: 31 aaggaaaaaa gcggccgcat tcatgttgag tag 33 <210> SEQ IDNO 32 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer 1896-56 <400> SEQUENCE: 32 acgcgtcgactcatcaattc atgttgagta gtttg 35 <210> SEQ ID NO 33 <211> LENGTH: 39 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: PCR primer 1896-57<400> SEQUENCE: 33 aaggaaaaaa gcggccgctc atcaattcat gttgagtag 39 <210>SEQ ID NO 34 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer 1954-45 <400> SEQUENCE: 34 acgcgtcgacttattatttc ctcctgaata g 31 <210> SEQ ID NO 35 <211> LENGTH: 42 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: PCR primer 1954-46<400> SEQUENCE: 35 aaggaaaaaa gcggccgctt attatttcct cctgaataga gc 42<210> SEQ ID NO 36 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: PCR primer 1955-44 <400> SEQUENCE: 36 cccaagcttaccatgagcac caaacctgat atg 33 <210> SEQ ID NO 37 <211> LENGTH: 34 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: PCR primer 1954-47<400> SEQUENCE: 37 cccaagctta ccatgattca aaagtgtttg tggc 34 <210> SEQ IDNO 38 <211> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer 1954-48 <400> SEQUENCE: 38 aaggaaaaaagcggccgcgc ggccctcgat tttcctcctg aatagagctg taa 53 <210> SEQ ID NO 39<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: PCR primer 1954-49 <400> SEQUENCE: 39 aaggaaaaaa gcggccgctttcctcctgaa tagagctgta a 41

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and (b)the amino acid sequence encoded by the DNA insert in ATCC Deposit No.PTA-976.
 2. An isolated polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asset forth in either SEQ ID NO: 3 or SEQ ID NO: 6, optionally furthercomprising an amino-terminal methionine; (b) an amino acid sequence foran ortholog of either SEQ ID NO: 2 or SEQ ID NO:5; (c) an amino acidsequence that is at least about 70 percent identical to the amino acidsequence of either SEQ ID NO: 2 or SEQ ID NO: 5, wherein the polypeptidehas an activity of the polypeptide set forth in either SEQ ID NO: 2 orSEQ ID NO: 5; (d) a fragment of the amino acid sequence set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 comprising at least about 25 aminoacid residues, wherein the fragment has an activity of the polypeptideset forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic; and(e) an amino acid sequence for an allelic variant or splice variant ofthe amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ IDNO: 5, the amino acid sequence encoded by the DNA insert in ATCC DepositNo. PTA-976, or the amino acid sequence of any of (a)-(c).
 3. Anisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of: (a) the amino acid sequence as set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acidsubstitution, wherein the polypeptide has an activity of the polypeptideset forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) the amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with atleast one amino acid insertion, wherein the polypeptide has an activityof the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (c)the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ IDNO: 5 with at least one amino acid deletion, wherein the polypeptide hasan activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQID NO: 5; (d) the amino acid sequence as set forth in either SEQ ID NO:2 or SEQ ID NO: 5 that has a C- and/or N-terminal truncation, whereinthe polypeptide has an activity of the polypeptide set forth in eitherSEQ ID NO: 2 or SEQ ID NO: 5; and (e) the amino acid sequence as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least onemodification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO:
 5. 4. An isolated polypeptide encoded by a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQID NO: 4; (b) the nucleotide sequence of the DNA insert in ATCC DepositNo. PTA-976; (c) a nucleotide sequence encoding the polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5; and (d) a nucleotidesequence that hybridizes under at least moderately stringent conditionsto the complement of any of (a)-(c); wherein the polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO:
 5. 5. An isolated polypeptide encoded by a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding a polypeptide that is at least about70 percent identical to the polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5; (b) a nucleotide sequence encoding an allelicvariant or splice variant of the nucleotide sequence as set forth ineither SEQ ID NO: 1 or SEQ ID NO: 4, the nucleotide sequence of the DNAinsert in ATCC Deposit No. PTA-976, or the nucleotide sequence of (a);(c) a region of the nucleotide sequence of either SEQ ID NO: 1 or SEQ IDNO: 4, the DNA insert in ATCC Deposit No. PTA-976, or the nucleotidesequence of (a) or (b), encoding a polypeptide fragment of at leastabout 25 amino acid residues; (d) a region of the nucleotide sequence ofeither SEQ ID NO: 1 or SEQ ID NO: 4, the DNA insert in ATCC Deposit No.PTA-976, or the nucleotide sequence of any of (a)-(c), comprising afragment of at least about 16 nucleotides; and (e) a nucleotide sequencethat hybridizes under at least moderately stringent conditions to thecomplement of any of (a)-(d); wherein the polypeptide has an activity ofthe polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO:
 5. 6. Anisolated polypeptide encoded by a nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5 with at least one conservative amino acidsubstitution; (b) a nucleotide sequence encoding a polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one aminoacid insertion; (c) a nucleotide sequence encoding a polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one aminoacid deletion; (d) a nucleotide sequence encoding a polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5 that has a C- and/orN-terminal truncation; (e) a nucleotide sequence encoding a polypeptideas set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least onemodification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation; (f) a nucleotide sequence of anyof (a)-(e) comprising a fragment of at least about 16 nucleotides; and(g) a nucleotide sequence that hybridizes under at least moderatelystringent conditions to the complement of any of (a)-(f); wherein thepolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO:
 5. 7. The isolated polypeptide according to claim2 or 3, wherein the percent identity is determined using a computerprogram selected from the group consisting of GAP, BLASTP, FASTA,BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm.
 8. Acomposition comprising the polypeptide of any of claims 1, 2, or 3, anda pharmaceutically acceptable formulation agent.
 9. The composition ofclaim 8, wherein the pharmaceutically acceptable formulation agent is acarrier, adjuvant, solubilizer, stabilizer, or anti-oxidant.
 10. Thecomposition of claim 8, wherein the polypeptide comprises the amino acidsequence as set forth in either SEQ ID NO: 3 or SEQ ID NO:
 6. 11. Apolypeptide comprising a derivative of the polypeptide of any of claims1, 2, or
 3. 12. The polypeptide of claim 11 that is covalently modifiedwith a water-soluble polymer.
 13. The polypeptide of claim 12, whereinthe water-soluble polymer is selected from the group consisting ofpolyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 14. A fusionpolypeptide comprising the polypeptide of any of claims 1, 2, or 3 fusedto a heterologous amino acid sequence.
 15. The fusion polypeptide ofclaim 14, wherein the heterologous amino acid sequence is an IgGconstant domain or fragment thereof.
 16. A polypeptide produced by aprocess comprising culturing a host cell comprising a vector comprisinga nucleic acid molecule comprising a nucleotide sequence selected fromthe group consisting of: (a) the nucleotide sequence as set forth ineither SEQ ID NO: 1 or SEQ ID NO: 4; (b) the nucleotide sequence of theDNA insert in ATCC Deposit No. PTA-976; (c) a nucleotide sequenceencoding the polypeptide as set forth in either SEQ ID NO: 2 or SEQ IDNO: 5; and (d) a nucleotide sequence that hybridizes under at leastmoderately stringent conditions to the complement of any of (a)-(c);under suitable conditions to express the polypeptide, and optionallyisolating the polypeptide from the culture.
 17. A polypeptide producedby a process comprising culturing a host cell comprising a vectorcomprising a nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a polypeptide that is at least about 70 percent identical tothe polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) a nucleotide sequenceencoding an allelic variant or splice variant of the nucleotide sequenceas set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, the nucleotidesequence of the DNA insert in ATCC Deposit No. PTA-976, or thenucleotide sequence of (a); (c) a region of the nucleotide sequence ofeither SEQ ID NO: 1 or SEQ ID NO: 4, the DNA insert in ATCC Deposit No.PTA-976, the nucleotide sequence (a) or (b), encoding a polypeptidefragment of at least about 25 amino acid residues, wherein thepolypeptide fragment has an activity of the encoded polypeptide as setforth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic; (d) aregion of the nucleotide sequence of either SEQ ID NO: 1 or SEQ ID NO:4, the DNA insert in ATCC Deposit No. PTA-976, or the nucleotidesequence of any of (a)-(c), comprising a fragment of at least about 16nucleotides; and (e) a nucleotide sequence that hybridizes under atleast moderately stringent conditions to the complement of any of(a)-(d); under suitable conditions to express the polypeptide, andoptionally isolating the polypeptide from the culture.
 18. A polypeptideproduced by a process comprising culturing a host cell comprising avector comprising a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide as set forth in either SEQ ID NO: 2 orSEQ ID NO: 5 with at least one conservative amino acid substitution,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) a nucleotide sequenceencoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO:5 with at least one amino acid insertion, wherein the encodedpolypeptide has an activity of the polypeptide set forth in either SEQID NO: 2 or SEQ ID NO: 5; (c) a nucleotide sequence encoding apolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with atleast one amino acid deletion, wherein the encoded polypeptide has anactivity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ IDNO: 5; (d) a nucleotide sequence encoding a polypeptide as set forth ineither SEQ ID NO: 2 or SEQ ID NO: 5 that has a C- and/or N-terminaltruncation, wherein the encoded polypeptide has an activity of thepolypeptide set forth in either SEQ ID NO:2or SEQ ID NO: 5; (e) anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 5 with at least one modification selected from thegroup consisting of amino acid substitutions, amino acid insertions,amino acid deletions, C-terminal truncation, and N-terminal truncation,wherein the encoded polypeptide has an activity of the polypeptide setforth in either SEQ ID NO: 2 or SEQ ID NO: 5; (f) a nucleotide sequenceof any of (a)-(e) comprising a fragment of at least about 16nucleotides; and (g) a nucleotide sequence that hybridizes under atleast moderately stringent conditions to the complement of any of(a)-(f); under suitable conditions to express the polypeptide, andoptionally isolating the polypeptide from the culture.
 19. Thepolypeptide of any of claims 16, 17, or 18, wherein the host cell is aeukaryotic cell.
 20. The polypeptide of any of claims 16, 17, or 18,wherein the host cell is a prokaryotic cell.